Methods of preventing secondary infections

ABSTRACT

A method of treating or preventing a disease associated with a secondary infection in a subject infected with a pathogen is provided. The method comprises administering to the subject a therapeutically effective amount of an anti-pathogenic agent directed towards the pathogen and a therapeutically effective amount of an agent which down-regulates at least one extracellular matrix-associated polypeptide.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/549,161 filed on Aug. 6, 2017, which is a National Phase of PCTPatent Application No. PCT/IL2016/050156 having International FilingDate of Feb. 9, 2016, which claims the benefit of priority under 35 USC§ 119(e) of U.S. Provisional Patent Application No. 62/113,551 filed onFeb. 9, 2015. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 82225SequenceListing.txt, created on Mar. 26,2020, comprising 121,355 bytes, submitted concurrently with the filingof this application is incorporated herein by reference. The sequencelisting submitted herewith is identical to the sequence listing formingpart of the international application.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to a methodof preventing secondary infections in subjects infected with a pathogenusing agents that down-regulate extracellular matrix remodeling.

Viral pandemics, such as influenza have caused millions of deathsworldwide. An extreme example is the 1918 pandemic which spread to sixcontinents and infected ˜500 million people reaching death toll of 50million. Investigation of clinical cases and autopsy samples indicatedthat more than 95% of case fatalities were complicated by secondarybacterial infections, most commonly Streptococcus pneumoniae (S.pneumoniae). Immune cells recruited to the site of infection arecritical for influenza clearance. However, growing evidence shows thatinfiltrating immune cells can also generate excessive inflammatoryresponses resulting in collateral tissue damage and disruption of theblood-air-barrier.

Tissue tolerance to pathogens is an important evolutionary trade-off,balancing the host immune response to pathogens while maintaining tissuefunction. However, tolerance capacity differs between various organs;lungs have a relatively low tissue tolerance capacity, and are morevulnerable to tissue damage. Accordingly, it has been argued that duringrespiratory viral infections uncontrolled host-derived immune responses,rather than viral titers, may be the leading cause of death. Theseresponses are primarily associated with inflammatory monocytes,granulocytes, macrophages and dendritic cells. Accordingly,influenza-infected lungs are diffusely hemorrhagic, potentially linkingthe host response with tissue destruction. Tissue breaching may primesecondary bacterial invasion coupled with tissue disruption and, inextreme cases, may result in death. The interaction between influenzaand secondary bacterial infections has long been studied, yet themolecular mechanisms by which influenza infection primes the tissue tosecondary infections are not fully understood.

One of the host's tolerance components is the integrity of respiratoryepithelial barriers anchored to the extracellular matrix (ECM). The ECMscaffold is produced by the cells in the tissue and is composed of twolayers: I) the interstitial matrix, a three-dimensional gel ofpolysaccharides and fibrous proteins, and II) the basement membrane, amesh-like sheet formed at the base of epithelial tissues. ECM turnoveris regulated by multiple proteolytic enzymes including matrixmetalloproteinases (MMPs) that are responsible for the irreversiblecleavage of a plethora of ECM molecules under normal and pathologicalconditions. Dysregulated proteolytic activity is often associated withinflammation, cancer, and infectious diseases. Accordingly, studies inpathological conditions have shown that dysregulated proteolysis of ECMmolecules and related protein fibers have significant effects on tissuefunction. Specifically, MMPs were shown to play critical roles in lungorganogenesis and many MMPs are involved in the acute and chronic phasesof lung inflammatory diseases (Greenlee et al., 2007, Physiologicalreviews 87, 69-98). Several substrates of MMPs have been identifiedduring lung development, including ECM scaffold proteins, cell adhesionmolecules, growth factors, cytokines, and chemokines (Greenlee et al.,2007, Physiological reviews 87, 69-98).

Membrane type-I matrix metalloproteinase (MT1-MMP/MMP-14), a membranetethered collagenase, is a key regulator in development and homeostasisof the lung as well as mediating wound healing, airway remodeling, andcell trafficking. Accordingly, it is expressed by multiple cellpopulations in the respiratory tract, including fibroblasts, endothelialcells and macrophages (Greenlee et al., 2007, Physiological reviews 87,69-98). The functions of macrophage-derived proteases duringinflammation are typically associated with tissue invasion ordegradative events. In macrophages MT1-MMP serves not only as a proteaseacting on the ECM, but also regulates macrophage immune response.Recruited monocytes and macrophages up-regulate a broad spectrum of ECMremodelers including various MMPs. Depending on the conditions,macrophages express a spectrum of MMPs and their inhibitors: these havebeen associated with both physiological and pathological lung remodelingevents. MMP-9 (gelatinase B) was shown to be beneficial for recoveryfrom influenza infection by promoting migration of neutrophils to theinfection site (Bradley et al., 2012, PLoS pathogens 8, e1002641).Despite these important findings, a systematic analysis of ECMproteolytic pathways during respiratory infections, including thetrade-off between ECM integrity and immune protection, has never beencompleted.

Background art includes Cheung et al., Cardiovasc Pathol. 2006March-April; 15(2):63-74, Elkington et al., 2005 British Society forImmunology, Clinical and Experimental Immunology, 142:12-20; Devy etal., Biochemistry Research International, Volume 2011, Article ID191670, doi:10.1155/2011/191670; Renckens et al., J Immunol 2006;176:3735-3741; Vanlaere et al., Clinical Microbiology Reviews, April2009, Vol 22, p. 224-239 and Udi et al., 2015, Structure 23, 1-12, Jan.6, 2015.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of treating or preventing a diseaseassociated with a secondary infection in a subject infected with apathogen comprising administering to the subject a therapeuticallyeffective amount of an anti-pathogenic agent directed towards thepathogen and a therapeutically effective amount of an agent whichdown-regulates at least one extracellular matrix-associated polypeptide,thereby treating or preventing the disease associated with a secondaryinfection in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a subject infected with apathogen comprising administering to the subject a therapeuticallyeffective amount of an anti-pathogenic agent directed towards thepathogen and a therapeutically effective amount of an agent whichdown-regulates at least one extracellular matrix-associated polypeptide,thereby treating the subject.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture comprising ananti-pathogenic agent and an agent which down-regulates at least oneextracellular matrix-associated polypeptide.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising ananti-pathogenic agent as a first active agent, an agent whichdown-regulates at least one extracellular matrix-associated polypeptideas a second active agent and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating influenza in a subject in needthereof comprising administering to the subject a therapeuticallyeffective amount of an agent which down-regulates an extracellularmatrix-associated polypeptide, thereby treating the influenza.

According to some embodiments of the invention, the extracellularmatrix-associated polypeptide is set forth in Table 1.

According to some embodiments of the invention, the secondary infectionis a bacterial infection, a viral infection or a fungal infection.

According to some embodiments of the invention, the secondary infectionis a blood infection.

According to some embodiments of the invention, the disease is sepsis.

According to some embodiments of the invention, the administeringcomprises co-administering.

According to some embodiments of the invention, the pathogen is selectedfrom the group consisting of a virus, a bacteria and a fungus.

According to some embodiments of the invention, the at least onepolypeptide is a matrix metalloproteinase (MMP).

According to some embodiments of the invention, the matrixmetalloproteinase is selected from the group consisting of membrane type1-matrix metalloproteinase 1 (MT1-MMP1), MMP-9, MMP-8 and MMP-3.

According to some embodiments of the invention, the at least onepolypeptide is membrane type 1-matrix metalloproteinase 1 (MT1-MMP1).

According to some embodiments of the invention, the infection is arespiratory infection.

According to some embodiments of the invention, the pathogen is a virus.

According to some embodiments of the invention, the virus is arespiratory virus.

According to some embodiments of the invention, the respiratory virus isinfluenza.

According to some embodiments of the invention, the anti-pathogenicagent is a neuraminidase inhibitor (NAI).

According to some embodiments of the invention, the neuraminidaseinhibitor is selected from the group consisting of Laninamivir,Oseltamivir, Peramivir and Zanamivir.

According to some embodiments of the invention, the neuraminidaseinhibitor is Oseltamivir.

According to some embodiments of the invention, the secondary infectionis a bacterial infection.

According to some embodiments of the invention, the bacterial infectionis S. pneumoniae.

According to some embodiments of the invention, the agent whichdown-regulates the at least one polypeptide is an antibody.

According to some embodiments of the invention, the agent whichdown-regulates the at least one polypeptide is a polynucleotide agent.

According to some embodiments of the invention, the extracellularmatrix-associated polypeptide is set forth in Table 1.

According to some embodiments of the invention, the at least onepolypeptide is a matrix metalloproteinase (MMP).

According to some embodiments of the invention, the matrixmetalloproteinase is selected from the group consisting of membrane type1-matrix metalloproteinase 1 (MT1-MMP1), MMP-9, MMP-8 and MMP-3.

According to some embodiments of the invention, the at least onepolypeptide is membrane type 1-matrix metalloproteinase 1 (MT1-MMP1).

According to some embodiments of the invention, the anti-pathogenicagent is an antiviral agent.

According to some embodiments of the invention, the anti-viral agent isa neuraminidase inhibitor (NAI).

According to some embodiments of the invention, the neuraminidaseinhibitor is selected from the group consisting of Laninamivir,Oseltamivir, Peramivir and Zanamivir.

According to some embodiments of the invention, the neuraminidaseinhibitor is Oseltamivir.

According to some embodiments of the invention, the extracellularmatrix-associated polypeptide is set forth in Table 1.

According to some embodiments of the invention, the extracellular matrixassociated polypeptide is MT1-MMP1.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-1D. Global analysis of extra cellular matrix gene circuitsduring influenza viral infection (A) K-means clustering (k=20) of 3530differentially expressed genes (Experimental Procedures) in lungsfollowing influenza infections at 10 time points (n=4 for each timepoint). Dynamic range is scaled between −2 to 2 fold changes and colorcoded. 13.5% (479) of the elevated genes are annotated as involved inECM remodeling. Functional annotation was done using(cbl-gorilladotcsdottechniondotacdotil) clusters are annotatedaccordingly and colored. (B) Shown are a subset of gene ontologies (GO)enriched (p<10-4) in infected lungs. (C) Submatrix of gene expressiondynamics following influenza infection of ECM remodeling genes. (D) Bargraph showing fold changes relative to T0 using qPCR measurement ofMT1-MMP expression following influenza infection. Each sample was run intriplicates from 4 mice (2 biological repeats). Error bars representstandard deviation (SD) of the average number. The target genes werenormalized to the endogenous reference gene GAPDH and relative to anon-infected control sample using ΔΔ CT normalization method.

FIGS. 2A-2C. MT1-MMP expression is mostly induced in myeloid cellsfollowing influenza infection. (A) FACS analysis from lung of influenzainfected mice 74 hours post viral infection (Experimental procedures;n=25) compared to non-infected controls (n=25). Gated are MT1-MMPexpressing cells stained using anti-MT1-MMP antibody as well as CD45,CD11b and Epcam (Experimental procedures). (B) Histogram plots showingMT1-MMP, Epcam and CD11b mean fluorescence intensity before (grey) and74 hours post influenza viral infection (red). (C) Bar graph showingqPCR measurement of MT1-MMP expression in sorted cell populations. Errorbars represent SD of the average number. T-test**p<0.001.

FIGS. 3A-3I. Influenza infection induces changes in ECM morphology (A)Global mass spectrometry analysis of cell free ECM scaffolds (seeexperimental procedures). Quantitative protein abundance is presented byrelative measurement (with reference to control uninfected tissue) usinggray scale color code ranging from −1 to 1 white to black. Proteinsdepleted from the ECM post infection are annotated and colored white.Heatmap showing significant changes in protein quantification (p<0.01,t-test) in de-cellularized infected lung tissue as compared tonon-infected control. Samples were analyzed in duplicates for 2 timepoints post infection (74, 122 hours post infection) with lethal dose ofinfluenza infection using Mascot software (B) Representative scanningelectron microscope imaging of infected versus control lungs. Arrows andarrow-heads point to orientation changes in collagen fibrils withD-banding patterns as quantified in sub figure (C) Directionality offibers on the boundaries of alveoli are analyzed using Fiji package(Experimental procedures). (D-H) Representative immuno-staining imagesof ECM components during infection taken from (n=20) animals andscreened in multiple tissue sections and slides imaged under the sameexposure conditions. (E-I) Quantification of immunostaining using imageJpackage (Experimental procedures), Error bars represent SD of theaverage number.

FIGS. 4A-4K. Blocking MT1-MMP activity protects lung ECM components. (A)Cartoon showing experimental setup for influenza infection with varioustreatments. Mice were infected with sub-lethal dose of PR8 influenzastrain (Experimental Procedures) (B-E) AirSEM imaging of alveolar andbronchial compartments 74 hours post infection using fixed tissuesections from whole lung, cut 300 μm thick and stained for AirSEM(Experimental Procedures). Alveolar wall thickness and bronchial cellnumbers were measured at different areas in multiple sections. Scale ofmain image-50 μm, inset scale—20 μm. Bar graph quantifies wall thicknessand cell numbers in alveoli and bronchi, respectively. Error barsrepresent SD of the average number; field of view (FOV). (F-G)Representative second harmonic generation (SHG) images originating froman unstained 50 μm thick lung tissue sections. The detected SHG signalrepresenting collagen is shown in red after reproduction of the z-stackusing Imaris software package version 7.7.1. Bar graph is showingcollagen volumes analyzed and quantified using Imaris package and testedfor significance using t-test. Error bars represent SD of the averagenumber (H-I) Lung immuno-staining for laminin. Bar graph shows lamininintensity analyzed using ImageJ package. (J-K) Collagen type I in situzymography in lung tissue using fluorogenic substrate to detectcollagenolytic activity in lung section with high sensitivity. Greensignal and arrows point to active collagenase localization amongbronchial epithelial lining cells or infiltrating immune cells. Scale—50μm; Error bars represent SD of the average number; field of view (FOV).

FIGS. 5A-5H. Combining anti-viral treatment with ECM protection supportssurvival and prevents systemic bacterial sepsis. (A, D) Cartoon showingexperimental setup for influenza and S. pneumoniae co-infections inpreventative and therapeutic modes. Mice were infected with sub-lethaldoses of influenza followed by infection with Strep. pneumoniae(Experimental procedures). Treatment groups included: Tamiflu,anti-MT1-MMP Fab, or the combinations of both. Administration was doneusing preventive mode, one day before infection (A-C) or as therapeuticmode one-day post infection (D-E). Vehicle-treated mice served ascontrols (Data are combined from three independent experiments with 7-10mice in each group). (B, E) Survival curves (Kaplan-Meier) ofco-infected mice receiving different treatments a day before (−1) or aday after (+1) the infections. Data is collected from 3 independentexperiments of 5 mice in each group. * P<0.01; **p<0.001 using Log-rank(Mantel-Cox) test. (C, F) Relative weight loss of co-infected mice atseveral time points post viral infection. Error bars represent SD fromthe mean. (G-H) S. pneumonia bacterial loads from spleen lysates ofinfected mice 6 days post viral infection (Experimental procedures).

FIGS. 6A-6D. Gene and protein expression levels of ECM modulators duringthe course of influenza infection. (A) Bar graph showing qPCRmeasurements of ECM representative genes during different time pointspost infection in whole lung tissue (fold change relative to expressionlevels in T0) infection with lethal dose of influenza infection(experimental procedures). Each sample was run in triplicates from 4mice (2 biological repeats). Error bars represent standard deviation(SD) (B) Western blot analysis of several representative proteasesduring different time points of influenza infection using a reducingSDS-PAGE gel. (n=10). (C) Quantification of western blot results usingImageJ software. Average relative density of the protein of interest isrelative to GAPDH internal control (D) Mean values of body weight (blue;left y axis) and viral titers (red; right y axis) during the course ofinfluenza infection. Error bars represent standard deviations of bodyweight, calculated on 2-4 animals in each time point from 3 independentexperiments. Viral burdens of whole lung homogenates in the lungs ofmice using qPCR for genome copies of Matrix protein 2 (M2) followed byconversion into viral particle numbers using a calibration curve.

FIGS. 7A-7D. Immune cells express active MT1-MMP during infection. (A)Immunostaining and bar graph quantification of MT1-MMP and F4/80 markerco-localization in infected lungs (74 hours PI) versus healthy controls.Arrows point to MT1-MMP stained cells. Representative images frommultiple sections. (B) Bar graph quantifying panel A. Error barrepresent SD, *P≤0.01, t-test. (C) Collagen type I in situ zymographycombined with CD45 staining. Arrows point to cells expressing eithermarker at both control and infected sections (74 hours PI). Scale bar-50μm (D) Bar graph quantifying panel C. Error bar represent SD, *P≤0.01,t-test.

FIG. 8. Global expression analysis of MT1-MMP expressing cells. (A)K-means clustering (k=6) of 2169 differentially expressed genes inCD45^(pos) and CD45^(neg) populations of cells sorted 74 hours postinfection (n≥5 mice included in each group-infected and non-infectedcontrol). Mice were infected with lethal dose of 4×10³ PFU of PR8influenza (experimental procedures).

FIGS. 9A-9D. Lung destructive phenotypes demonstrated using AirSEMimaging of whole lung or de-cellularized tissue. (A) Imaging of wholelung tissue. Arrows point to boundaries of alveolar openings with cells(non-infected control) or depleted of cells (infected) (B) Imaging ofECM scaffolds (after de-cellularization) of infected lungs compared tohealthy controls. Arrows point to alveolar duct boundaries containingthick organized collagen bundles (non-infected control) or distortedfibrils (infected). (C) Lung cell counts in control and infected lungsscanning multiple lung sections, n=5. (D) Directionality imaginganalysis was done by Fiji package. Graphs were plotted using GraphPadPrism 6. The relative frequency of fiber spatial orientation wasmeasured using the “Directionality” plugin analysis tool in Fiji packageversion 6.1.1.

FIGS. 10A-10C. Calibration of single viral infection. (A) Weight loss ofmice subjected to single viral infection at different dosages. Data setwas analyzed from 5 mice at each time point. Error bar represent SD andanalyzed using t-test. (B) Survival of mice exposed to single viralinfection at different dosages. Error bar represent SD and analyzedusing t-test**P≤0.001. (C) Viral burdens of whole lung homogenates inthe lungs 4 days post infection using standard PFU assay. Samples wererun with 2 biological repeats 3 animals at each time point. X-axisrepresents the viral amounts used for the infection.

FIGS. 11A-11F. MT1-MMP inhibition does not interfere with immune cellrecruitment or cytokine induction. (A) FACS analysis of whole lungtissue subjected to influenza infection 74 hours post infection andtreated either with anti-MT1-MMP inhibitor antibody or non-relevant GSTcontrol Ab. Mice were infected at sub-lethal influenza dose(experimental procedures). Data is gated on MT1-MMP expressing cellsstained with MT1-MMP antibody as well as CD45, Ly6G, Ly6C, CD11b, NK46,TCRβ (Experimental procedures). Experiments were done twice using 3 miceper group. (B) Representative sections of lung tissue stained formacrophages using F4/80 marker taken at 74 hours post infection. Scalebars=50 μm. FOV indicates the entire field of view at magnification of×20. (C) Quantification of figure B using multiple tissue sections fromat least 3 mice per group. Error bar represent SD, ***P≤0.0001, t-test.(D) Infiltrating immune cells in BALF from mice subjected to singleviral infection and taken at 24, 48, 72, 122 hours PI. Mice wereinfected with sub-lethal dose of influenza. Samples were run with 2biological repeats. Tested significant over non-infected mice usingt-test*P≤0.01. (E-F) TNF-α and IL-1β levels in BALF of mice subjected tosingle viral infection and taken at 24, 48, 72, 96, 122 hours PI.Samples were run with 2 biological repeats. Error bar represent SD andanalyzed using t-test*P≤0.01.

FIGS. 12A-12F. Viral loads in the lung following Anti-MT1-MMP Abtreatment 74 Hours PI (A) Representative lung tissue sections stainedfor influenza virus using Tamiflu, control Ab and anti-MT1-MMP Ab. Micewere infected sub-lethal dose of influenza (experimental procedures).(B-C) Bar graph quantification of influenza virus 24 and 48 hours PI.Error bar represent SD, **P≤0.001, t-test. FOV indicates the entirefield of view at magnification of ×20. Number of infected cells wasnormalized to DAPI using ImageJ. (D) PFU values of whole lung tissue 4days and 7 days post viral infection (experimental procedures). Sampleswere run in triplicates of 2 biological repeats. Error bar represent SD.LEM −1; Tami-1; Tami+LEM-1 designate the different treatments, single orcombined agents, given one day before the infection (Day-1). LEM+1;Tami+1; Tami+LEM+1 designate the different treatments, single orcombined agents, given one day after the infection (Day+1). LEM refersto anti-MT1-MMP Ab (LEM2/15), GST refers to non-relevant Ab. (E) Viralburdens in the lungs 24, 48 and 96 hours post infection. Whole lunghomogenates were used for PFU assay, testing 2 animals at each timepoint and running 2 biological replicates. Error bar represent SD,*P≤0.01 using 2-way ANOVA. (F) CFU values in the lungs of co-infectedmice 2 days post bacterial infection. Error bars represent SD from themean. Data are combined from two independent experiments with five micein each group.

FIGS. 13A-13D. ECM destruction is not perturbed by low viral titers. (A)AirSEM images of lungs 74 hours post infection from eitherTamiflu-treated, vehicle-treated or control mice. (B) Alveolar wallthickness measured using ImageJ (Experimental procedures), each markrepresents a mean of measurements from a section-based region for anindividual animal. (C) Bar graph represents viral counts invehicle-treated and Tamiflu-treated mice lungs using qPCR. Each columnrepresents the mean of 3 mice. Bars indicate mean and SD from theaverage. X-axis represents hours post viral infection. (D) MT1-MMPexpression levels in mice lungs infected with 90 PFU of PR8 influenzastrain undergoing different treatments. Error bar represent SD andanalyzed using t-test**P≤0.001.

FIGS. 14A-14B. Combined anti-viral and tissue protection therapymaintains lung structural features. (A) AirSEM imaging of lung sectionsrepresenting changes in lung bronchi and alveoli during infection underseveral treatment modalities. Scale bar −20 μm. (B) Bar graphquantifying cell numbers in the different treatments taken from multiplesections.

FIG. 15. MT1-MMP expression in human respiratory epithelial cells uponinfluenza infection. Log2 relative expression levels of MT1-MMPcorrelating with the infection course (hours post infection) of humanbronchial epithelial cells infected with H1N1 strain A/PR/8/34 (PR8).Error bars represent SD. Data analyzed from (Shapira S D, 2009).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a methodof preventing secondary infections in subjects infected with a pathogenusing agents that down-regulate extracellular matrix remodeling.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Infectious disease treatments have conventionally focused on pathogenelimination, either by administering antimicrobial drugs or bystimulating host immune responses using vaccination. The presentinventors performed global genomics and proteomics analyses of aninfluenza mouse model and revealed an unexpected plethora ofextracellular matrix (ECM)-related genes and proteins responsible fordysregulated ECM remodeling events during the course of infection (FIGS.1A-1D and 6A-6D). MT1-MMP was the main collagenase leading todestruction of ECM scaffolds of alveoli and bronchi of infected mouselungs. Electron microscopy of intact lungs, global mass spectrometry,two-photon and immune staining, and tissue zymography, revealed amultifaceted destruction of basement membrane components (FIGS. 3A-3Iand 9A-9D). This unprecedented damage to lungs contributed to loss ofblood-air barrier and resulted in systemic spread of secondary bacterialinfection through leakage from lungs to internal organs causing sepsisand mortality. These devastating phenotypes and resulting deadly outcomewere reversed by blocking the activity of MT1-MMP (FIGS. 4A-4K), thusoffering a new mode of therapeutic intervention through tissue support.As shown in FIGS. 5A-5H, combining anti-viral treatment with ECMprotection supports survival and prevents systemic bacterial sepsis.

The present inventors suggest this novel treatment opportunity forinfection, designed to support tissue morphology and homeostasis whilemitigating inappropriate host responses and collateral tissue damage.

Thus, according to a first aspect of the present invention there isprovided a method of treating a subject infected with a pathogencomprising administering to the subject a therapeutically effectiveamount of an anti-pathogenic agent directed towards the pathogen and atherapeutically effective amount of an agent which down-regulates atleast one extracellular matrix-associated polypeptide, herein below,thereby treating the subject.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

As used herein, the term “subject” refers to a mammalian subject—forexample a human subject.

The subjects who are treated have pathogens which cause an infection.

As used herein, the term “pathogen” refers to a microbe or microorganismsuch as a virus, bacterium, prion or fungus that causes a disease (e.g.a respiratory disease).

According to a particular embodiment, the pathogen is a human pathogen.

Exemplary pathogenic viruses may belong to the following families:Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae,Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae,Papovaviridae, Polyomavirus, Rhabdoviridae, Togaviridae. Particularpathogenic viruses contemplated by the present invention are those thatcause smallpox, influenza, mumps, measles, chickenpox, ebola, orrubella.

According to a particular embodiment, the virus is one which bringsabout a respiratory infection (e.g. an upper respiratory tract infectionand/or a lower respiratory tract infection).

Thus, according to a particular embodiment, the pathogenic virus is aninfluenza virus (e.g. influenza virus A—(e.g. H1N1, H2N2, H3N2, H5N1,H7N7, H1N2, H9N2, H7N2, H7N3, H10N7 and H7N9), influenza virus B orinfluenza virus C).

In another embodiment, the pathogenic virus is a parainfluenza virus(hPIV) including the human parainfluenza virus type 1 (hPIV-1) (causescroup); the human parainfluenza virus type 2 (hPIV-2) (causes croup andother upper and lower respiratory tract illnesses), the humanparainfluenza virus type 3 (hPIV-3) (associated with bronchiolitis andpneumonia) and the human parainfluenza virus type 4 (hPIV-4).

In yet another embodiment, the pathogenic virus is a respiratorysyncytial virus (RSV).

Exemplary pathogenic bacteria include Mycobacterium tuberculosis whichcauses tuberculosis, Streptococcus and Pseudomonas which causepneumonia, and Shigella, Campylobacter and Salmonella which causefoodborne illnesses. Other exemplary pathogenic bacteria contemplated bythe present invention are those that cause infections such as tetanus,typhoid fever, diphtheria, syphilis and Hansen's disease.

According to one embodiment, the pathogen causes an acute infection inthe subject.

According to another embodiment, the pathogen causes a chronic infectionin the subject.

The term “anti-pathogenic agent” refers to an antimicrobial agent andincludes, but is not limited to antiviral agents, antibacterial agents,antiviral agents, anti-prion agents.

I. Antiviral Agents

Antiviral agents which can be used for combination therapy according toaspects of the present invention include CRX4 and CCR5 receptorinhibitors such as amantadine and rimantadine and pleconaril. Furtherantiviral agents that can be used in the combination therapy of thisaspect of the present invention include agents which interfere withviral processes that synthesize virus components after a virus invades acell. Representative agents include nucleotide and nucleoside analoguesthat look like the building blocks of RNA or DNA, but deactivate theenzymes that synthesize the RNA or DNA once the analogue isincorporated. Acyclovir is a nucleoside analogue, and is effectiveagainst herpes virus infections. Zidovudine (AZT), 3TC, FTC, and othernucleoside reverse transcriptase inhibitors (NRTI), as well asnon-nucleoside reverse transcriptase inhibitors (NNRTI), can also beused. Integrase inhibitors can also be used. Other antiviral agentsinclude antisense oligonucleotides and ribozymes (directed against viralRNA or DNA at selected sites).

Some viruses, such as HIV, include protease enzymes, which cleave viralprotein chains apart so they can be assembled into their finalconfiguration. Protease inhibitors are another type of antiviral agentthat can be used in the combination therapy described herein.

The final stage in the life cycle of a virus is the release of completedviruses from the host cell. Some active agents, such as zanamivir(Relenza) and oseltamivir (Tamiflu) treat influenza by preventing therelease of viral particles by blocking a molecule named neuraminidasethat is found on the surface of flu viruses.

Still other antiviral agents function by stimulating the patient'simmune system. Interferons, including pegylated interferons, arerepresentative compounds of this class. Interferon alpha is used, forexample, to treat hepatitis B and C. Various antibodies, includingmonoclonal antibodies, can also be used to target viruses.

Anti-Bacterial Agents:

The antibacterial agent which can be used for combination therapyaccording to aspects of the present invention may be bactericidal orbacteriostatic.

In one embodiment, the antibacterial agent is an antibiotic.

As used herein, the term “antibiotic agent” refers to a group ofchemical substances, isolated from natural sources or derived fromantibiotic agents isolated from natural sources, having a capacity toinhibit growth of, or to destroy bacteria. Examples of antibiotic agentsinclude, but are not limited to; Amikacin; Amoxicillin; Ampicillin;Azithromycin; Azlocillin; Aztreonam; Aztreonam; Carbenicillin; Cefaclor;Cefepime; Cefetamet; Cefinetazole; Cefixime; Cefonicid; Cefoperazone;Cefotaxime; Cefotetan; Cefoxitin; Cefpodoxime; Cefprozil; Cefsulodin;Ceftazidime; Ceftizoxime; Ceftriaxone; Cefuroxime; Cephalexin;Cephalothin; Cethromycin; Chloramphenicol; Cinoxacin; Ciprofloxacin;Clarithromycin; Clindamycin; Cloxacillin; Co-amoxiclavuanate;Dalbavancin; Daptomycin; Dicloxacillin; Doxycycline; Enoxacin;Erythromycin estolate; Erythromycin ethyl succinate; Erythromycinglucoheptonate; Erythromycin lactobionate; Erythromycin stearate;Erythromycin; Fidaxomicin; Fleroxacin; Gentamicin; Imipenem; Kanamycin;Lomefloxacin; Loracarbef; Methicillin; Metronidazole; Mezlocillin;Minocycline; Mupirocin; Nafcillin; Nalidixic acid; Netilmicin;Nitrofurantoin; Norfloxacin; Ofloxacin; Oxacillin; Penicillin G;Piperacillin; Retapamulin; Rifaxamin, Rifampin; Roxithromycin;Streptomycin; Sulfamethoxazole; Teicoplanin; Tetracycline; Ticarcillin;Tigecycline; Tobramycin; Trimethoprim; Vancomycin; combinations ofPiperacillin and Tazobactam; and their various salts, acids, bases, andother derivatives. Anti-bacterial antibiotic agents include, but are notlimited to, aminoglycosides, carbacephems, carbapenems, cephalosporins,cephamycins, fluoroquinolones, glycopeptides, lincosamides, macrolides,monobactams, penicillins, quinolones, sulfonamides, and tetracyclines.

Antibacterial agents also include antibacterial peptides. Examplesinclude but are not limited to abaecin; andropin; apidaecins; bombinin;brevinins; buforin II; CAP18; cecropins; ceratotoxin; defensins;dermaseptin; dermcidin; drosomycin; esculentins; indolicidin; LL37;magainin; maximum H5; melittin; moricin; prophenin; protegrin; and ortachyplesins.

Anti-Fungal Agents:

The term “anti-fungal agent” refers to an agent or chemical thatinterferes with fungal infection through blocking spore germination,adhesion to substrates, or interfering with any metabolic process orstep that is required for growth and development of the fungus or itsspores.

Anti-Protozoal Agent:

The term “anti-protozoal” as used herein refers to any chemical or agentthat interferes with the parasitic or other life cycle features of abroad range of eukaryotic microbes and invertebrate worms. The agent orchemical might block protein synthesis, essential lipid production,respiratory processes or other metabolic events or growth control steps.

As mentioned herein above, the present invention contemplatesadministering both an agent directed against the pathogen (as detailedherein above) and an agent which down-regulates at least oneextracellular matrix-associated polypeptide.

The term one extracellular matrix-associated polypeptide refers to apolypeptide that reduces the formation or enhances the degradation ofthe extracellular matrix or is comprised in the extracellular matrix.

According to a particular embodiment, the extracellularmatrix-associated polypeptide is a fibrous protein such as collagen,elastin, fibronectin, and laminin.

According to another embodiment, the extracellular matrix-associatedpolypeptide is a protease such as a matrix metalloproteinase, an enzymebelonging to the class A Disintegrin And Metalloproteinase withThrombospondin Motifs (ADAMTS) including ADAMTS 1-17 and those belongingto the lysyl oxidase family such as Lysyl oxidase homolog 2 (LOXL).

In one embodiment, the extracellular matrix-associated polypeptide isset forth in Table 2B of the Examples section herein below.

Preferably, the extracellular matrix-associated polypeptide is set forthin Table 1, herein below. Exemplary cDNA sequences of each of the genesare provided therein.

TABLE 1 Symbol Gene (Human) SEQ ID Gene (mouse) TIMP1 NM_003254.2 1NM_001044384 ADAMTS4 NM_005099.4 2 NM_172845 TNC NM_002160.3 3 NM_011607VCAN NM_001126336.2 4 NM_001134475 THBS1 NM_003246.3 5 NM_011580 PLAUNM_001145031.1 6 NM_008873 HAS1 NM_001297436.1 7 NM_008215 SERPINA3NM_001085.4 8 NM_001033335 (3F), NM_009253 (3M), NM_009251 (3G)NM_009252 (3N) SERPINE1 NM_000602 9 NM_008871 MMP3 NM_002422.3 10NM_010809 ADAMTS15 NM_139055.2 11 NM_001024139 PRSS22 NM_022119.3 12NM_133731 ITGA5 NM_002205.2 13 NM_010577 LGMN NM_005606.6 14 NM_011175MMP14 NM_004995.3 15 NM_008608 GZMB NM_00413L4 16 NM_013542 MMP9NM_004994.2 17 NM_013599 LCN2 NM_005564.3 18 NM_008491 MMP8NM_001304441.1 19 NM_008611 LOXL3 NM_001289164.1 20 NM_013586 AIF1NM_001623.3 21 NM_019467 LOXL2 NM_002318.2 22 NM_033325 TIMP3NM_000362.4 23 NM_011595 LOXL1 NM_005576.3 24 NM_010729 ADAM8NM_001109.4 25 NM_007403 SERPING1 NM_000062.2 26 NM_009776 SERINC3NM_006811.2 27 NM_012032

Downregulation of ECM-associated polypeptides can be effected on thegenomic and/or the transcript level using a variety of molecules whichinterfere with transcription and/or translation [e.g., RNA silencingagents (e.g., antisense, siRNA, shRNA, microRNA), Ribozyme and DNAzyme],or on the protein level using e.g., antagonists, enzymes that cleave thepolypeptide and the like.

Following is a list of agents capable of downregulating expression leveland/or activity of ECM-associated polypeptides.

One example, of an agent capable of ECM-associated polypeptides is anantibody or antibody fragment capable of specifically binding theretoand down-regulating activity thereof.

Preferably, the antibody binds with a Ki of less than 1000 nm, morepreferably less than 100 nm and even more preferably less than 10 nm toits target polypeptide.

Preferably, the antibody specifically binds at least one epitope of thepolypeptide. As used herein, the term “epitope” refers to any antigenicdeterminant on an antigen to which the paratope of an antibody binds.

Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or carbohydrate side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics.

According to one embodiment, the epitopic determinant is on the surfaceof the polypeptide.

According to another embodiment, when the polypeptide is a matrixmetalloproteinase (MMP) such as MT1-MMP1, MMP-9, MMP-8 and MMP-3 theantibody binds to (and may optionally be generated by immunizing with) ahapten compound,[2-(2-minoethylcarbomoyl)-ethoxymethyl]-tris-[2-(N-(3-imidazol-1-yl-propyl))-ethoxymethyl]methane.This hapten molecule closely mimics the local structure and conformationof the reactive zinc site in MMPs (see WO 2008/102359, the contents ofwhich are incorporated herein by reference).

In one embodiment, the antibody is capable of specifically binding tothe active form of the antibody and not to the proenzyme form.

Preferably, the antibody is specific to the particular matrixmetalloproteinase (MMP) and binds with at least 5 times higher affinityto that particular MMP than a non relevant MMP.

According to a specific embodiment, the polypeptide is MT1-MMP1, alsoknown as MMP-14.

Examples of antibodies that bind and down-regulate MMP-14 include thoseproduced by the LEM-2/15 hybridoma cells as detailed in Udi et al.,Structure 23, 1-12, Jan. 6, 2015, the contents of which are incorporatedherein by reference.

According to another embodiment, the antibody targets a surface epitopeof MMP-14. Thus, for example the antibody may bind to the VB loop ofMMP-14 (for example residues 160-173 and/or residues 218-233 of MMP-14).In another embodiment, the antibody is one which causes a conformationalswiveling motion of the V-B loop of MMP-14.

An exemplary amino acid sequence of the V_(H) of a MMP-14 downregulatingantibody is presented in SEQ ID NO: 54. An exemplary amino acid sequenceof the V_(L) of a MMP-14 downregulating antibody is presented in SEQ IDNO: 55.

In yet another embodiment, the antibody is such that it down-regulatesthe collagenase activity of MMP-14, but does not affect the activationof pro-MMP-2.

Additional antibodies which down-regulate MMP-14 are disclosed in U.S.Pat. No. 8,501,181 and Devy et al., Biochemistry Research International,Volume 2011, Article ID 191670, 11 pages, doi:10.1155/2011/191670.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, and Fvthat are capable of binding to macrophages. These functional antibodyfragments are defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule, can beproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule that can be obtained by treating wholeantibody with pepsin, followed by reduction, to yield an intact lightchain and a portion of the heavy chain; two Fab′ fragments are obtainedper antibody molecule; (3) (Fab′)2, the fragment of the antibody thatcan be obtained by treating whole antibody with the enzyme pepsinwithout subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragmentsheld together by two disulfide bonds; (4) Fv, defined as a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains; and(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference).

Antibody fragments according to some embodiments of the invention can beprepared by proteolytic hydrolysis of the antibody or by expression inE. coli or mammalian cells (e.g. Chinese hamster ovary cell culture orother protein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc.Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by [Whitlow andFilpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426(1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No.4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues form acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

Down-regulation of ECM-associated polypeptides can be also achieved byRNA silencing. As used herein, the phrase “RNA silencing” refers to agroup of regulatory mechanisms [e.g. RNA interference (RNAi),transcriptional gene silencing (TGS), post-transcriptional genesilencing (PTGS), quelling, co-suppression, and translationalrepression] mediated by RNA molecules which result in the inhibition or“silencing” of the expression of a corresponding protein-coding gene.RNA silencing has been observed in many types of organisms, includingplants, animals, and fungi.

As used herein, the term “RNA silencing agent” refers to an RNA which iscapable of specifically inhibiting or “silencing” the expression of atarget gene. In certain embodiments, the RNA silencing agent is capableof preventing complete processing (e.g, the full translation and/orexpression) of an mRNA molecule through a post-transcriptional silencingmechanism. RNA silencing agents include noncoding RNA molecules, forexample RNA duplexes comprising paired strands, as well as precursorRNAs from which such small non-coding RNAs can be generated. ExemplaryRNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.In one embodiment, the RNA silencing agent is capable of inducing RNAinterference. In another embodiment, the RNA silencing agent is capableof mediating translational repression.

According to an embodiment of the invention, the RNA silencing agent isspecific to the target RNA (e.g., MMP-14) and does not cross inhibit orsilence a gene or a splice variant which exhibits 99% or less globalhomology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%,93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% globalhomology to the target gene.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs). The corresponding process in plants iscommonly referred to as post-transcriptional gene silencing or RNAsilencing and is also referred to as quelling in fungi. The process ofpost-transcriptional gene silencing is thought to be anevolutionarily-conserved cellular defense mechanism used to prevent theexpression of foreign genes and is commonly shared by diverse flora andphyla. Such protection from foreign gene expression may have evolved inresponse to the production of double-stranded RNAs (dsRNAs) derived fromviral infection or from the random integration of transposon elementsinto a host genome via a cellular response that specifically destroyshomologous single-stranded RNA or viral genomic RNA.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes. The RNAi response also features anendonuclease complex, commonly referred to as an RNA-induced silencingcomplex (RISC), which mediates cleavage of single-stranded RNA havingsequence complementary to the antisense strand of the siRNA duplex.Cleavage of the target RNA takes place in the middle of the regioncomplementary to the antisense strand of the siRNA duplex.

Accordingly, some embodiments of the invention contemplate use of dsRNAto down-regulate protein expression from mRNA.

According to one embodiment, the dsRNA is greater than 30 bp. The use oflong dsRNAs (i.e. dsRNA greater than 30 bp) has been very limited owingto the belief that these longer regions of double stranded RNA willresult in the induction of the interferon and PKR response. However, theuse of long dsRNAs can provide numerous advantages in that the cell canselect the optimal silencing sequence alleviating the need to testnumerous siRNAs; long dsRNAs will allow for silencing libraries to haveless complexity than would be necessary for siRNAs; and, perhaps mostimportantly, long dsRNA could prevent viral escape mutations when usedas therapeutics.

Various studies demonstrate that long dsRNAs can be used to silence geneexpression without inducing the stress response or causing significantoff-target effects—see for example [Strat et al., Nucleic AcidsResearch, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res.Protoc. 2004; 13:115-125; Diallo M., et al., Oligonucleotides. 2003;13:381-392; Paddison P. J., et al., Proc. Natl Acad. Sci. USA. 2002;99:1443-1448; Tran N., et al., FEBS Lett. 2004; 573:127-134].

In particular, the invention according to some embodiments thereofcontemplates introduction of long dsRNA (over 30 base transcripts) forgene silencing in cells where the interferon pathway is not activated(e.g. embryonic cells and oocytes) see for example Billy et al., PNAS2001, Vol 98, pages 14428-14433. and Diallo et al, Oligonucleotides,Oct. 1, 2003, 13(5): 381-392. doi:10.1089/154545703322617069.

The invention according to some embodiments thereof also contemplatesintroduction of long dsRNA specifically designed not to induce theinterferon and PKR pathways for down-regulating gene expression. Forexample, Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] havedeveloped a vector, named pDECAP, to express long double-strand RNA froman RNA polymerase II (Pol II) promoter. Because the transcripts frompDECAP lack both the 5′-cap structure and the 3′-poly(A) tail thatfacilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP doesnot induce the interferon response.

Another method of evading the interferon and PKR pathways in mammaliansystems is by introduction of small inhibitory RNAs (siRNAs) either viatransfection or endogenous expression.

The term “siRNA” refers to small inhibitory RNA duplexes (generallybetween 18-30 basepairs) that induce the RNA interference (RNAi)pathway. Typically, siRNAs are chemically synthesized as 21mers with acentral 19 bp duplex region and symmetric 2-base 3′-overhangs on thetermini, although it has been recently described that chemicallysynthesized RNA duplexes of 25-30 base length can have as much as a100-fold increase in potency compared with 21mers at the same location.The observed increased potency obtained using longer RNAs in triggeringRNAi is theorized to result from providing Dicer with a substrate(27mer) instead of a product (21mer) and that this improves the rate orefficiency of entry of the siRNA duplex into RISC.

It has been found that position of the 3′-overhang influences potency ofan siRNA and asymmetric duplexes having a 3′-overhang on the antisensestrand are generally more potent than those with the 3′-overhang on thesense strand (Rose et al., 2005). This can be attributed to asymmetricalstrand loading into RISC, as the opposite efficacy patterns are observedwhen targeting the antisense transcript.

The strands of a double-stranded interfering RNA (e.g., an siRNA) may beconnected to form a hairpin or stem-loop structure (e.g., an shRNA).Thus, as mentioned the RNA silencing agent of some embodiments of theinvention may also be a short hairpin RNA (shRNA).

The term “shRNA”, as used herein, refers to an RNA agent having astem-loop structure, comprising a first and second region ofcomplementary sequence, the degree of complementarity and orientation ofthe regions being sufficient such that base pairing occurs between theregions, the first and second regions being joined by a loop region, theloop resulting from a lack of base pairing between nucleotides (ornucleotide analogs) within the loop region. The number of nucleotides inthe loop is a number between and including 3 to 23, or 5 to 15, or 7 to13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can beinvolved in base-pair interactions with other nucleotides in the loop.It will be recognized by one of skill in the art that the resultingsingle chain oligonucleotide forms a stem-loop or hairpin structurecomprising a double-stranded region capable of interacting with the RNAimachinery.

It will be appreciated that the RNA silencing agent of some embodimentsof the invention need not be limited to those molecules containing onlyRNA, but further encompasses chemically-modified nucleotides andnon-nucleotides.

In some embodiments, the RNA silencing agent provided herein can befunctionally associated with a cell-penetrating peptide.” As usedherein, a “cell-penetrating peptide” is a peptide that comprises a short(about 12-30 residues) amino acid sequence or functional motif thatconfers the energy-independent (i.e., non-endocytotic) translocationproperties associated with transport of the membrane-permeable complexacross the plasma and/or nuclear membranes of a cell. Thecell-penetrating peptide used in the membrane-permeable complex of someembodiments of the invention preferably comprises at least onenon-functional cysteine residue, which is either free or derivatized toform a disulfide link with a double-stranded ribonucleic acid that hasbeen modified for such linkage. Representative amino acid motifsconferring such properties are listed in U.S. Pat. No. 6,348,185, thecontents of which are expressly incorporated herein by reference. Thecell-penetrating peptides of some embodiments of the inventionpreferably include, but are not limited to, penetratin, transportan,pIsl, TAT (48-60), pVEC, MTS, and MAP.

mRNAs to be targeted using RNA silencing agents include, but are notlimited to, those whose expression is correlated with an undesiredphenotypic trait. Exemplary mRNAs that may be targeted are those thatencode truncated proteins i.e. comprise deletions. Accordingly the RNAsilencing agent of some embodiments of the invention may be targeted toa bridging region on either side of the deletion. Introduction of suchRNA silencing agents into a cell would cause a down-regulation of themutated protein while leaving the non-mutated protein unaffected.

According to another embodiment the RNA silencing agent may be a miRNAor miRNA mimic.

The term “microRNA”, “miRNA”, and “miR” are synonymous and refer to acollection of non-coding single-stranded RNA molecules of about 19-28nucleotides in length, which regulate gene expression. miRNAs are foundin a wide range of organisms (viruses.fwdarw.humans) and have been shownto play a role in development, homeostasis, and disease etiology.

The term “microRNA mimic” refers to synthetic non-coding RNAs that arecapable of entering the RNAi pathway and regulating gene expression.miRNA mimics imitate the function of endogenous microRNAs (miRNAs) andcan be designed as mature, double stranded molecules or mimic precursors(e.g., or pre-miRNAs). miRNA mimics can be comprised of modified orunmodified RNA, DNA, RNA-DNA hybrids, or alternative nucleic acidchemistries (e.g., LNAs or 2′-O,4′-C-ethylene-bridged nucleic acids(ENA)). For mature, double stranded miRNA mimics, the length of theduplex region can vary between 13-33, 18-24 or 21-23 nucleotides. ThemiRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides. The sequence ofthe miRNA may be the first 13-33 nucleotides of the pre-miRNA. Thesequence of the miRNA may also be the last 13-33 nucleotides of thepre-miRNA.

Another agent capable of downregulating ECM-associated polypeptides is aDNAzyme molecule capable of specifically cleaving an mRNA transcript orDNA sequence of the polypeptide.

Downregulation ECM-associated polypeptides can also be effected by usingan antisense polynucleotide capable of specifically hybridizing with anmRNA transcript encoding same.

Another agent capable of downregulating ECM-associated polypeptides is aribozyme molecule capable of specifically cleaving an mRNA transcriptencoding same.

Another agent capable of downregulating ECM-associated polypeptideswould be any molecule which binds to and/or cleaves the polypeptide.Such molecules can be antagonists, or inhibitory peptide.

For example, Zarrabi et al (J Biol Chem. 2011 Sep. 23; 286(38):33167-33177) discloses peptides that inhibit MMP-14.

It will be appreciated that a non-functional analogue of at least acatalytic or binding portion of any of the disclosed polypeptides can bealso used as an agent which down-regulates ECM-associated polypeptides.

Another agent which can be used along with some embodiments of theinvention to down-regulate the ECM-associated polypeptides is a moleculewhich prevents activation or substrate binding thereto.

Additional exemplary inhibitors of matrix metalloproteinases include thehydroxamate inhibitors, small peptide analogs of fibrillar collagens,which specifically interact in a bidentate manner via the hydroxyl andcarbonyl oxygens of the hydroxamic group with the zinc ion in thecatalytic site [Grams et al., (1995), Biochem. 34: 14012-14020; Bode etal., (1994), EMBO J., 13: 1263-1269].

Hydroxamate-based MMP inhibitors are usually composed of either a carbonback-bone (WO 95/29892, WO 97/24117, WO 97/49679 and EP 0780386), apeptidyl back-bone (WO 90/05719, WO 93/20047, WO 95/09841 and WO96/06074) or a peptidomimetic back-bone [Schwartz et al., Progr. Med.Chem., 29: 271-334(1992); Rasmussen et al., Pharmacol. Ther., 75: 69-75(1997); Denis et al., Invest. New Drugs, 15: 175-185 (1997)].Alternatively, they contain a sulfonamido sulfonyl group which is bondedon one side to a phenyl ring and a sulfonamido nitrogen which is bondedto an hydroxamate group via a chain of one to four carbon atoms (EP0757984 A1).

Other peptide-based MMP inhibitors are thiol amides which exhibitcollagenase inhibition activity (U.S. Pat. No. 4,595,700),N-carboxyalkyl derivatives containing a biphenylethylglycine whichinhibit MMP-3, MMP-2 and collagenase (Durette, et al., WO-9529689),lactam derivatives which inhibit MMPs, TNF-alpha and aggrecanase (seeU.S. Pat. No. 6,495,699) and Tricyclic sulfonamide compounds (see U.S.Pat. No. 6,492,422).

Other MMP inhibitors are the chemically modified nonmicrobialtetracyclines (CMTs) that were shown to block expression of several MMPsin vitro. (Axisa et al., 2002, Stroke 33: 2858-2864).

Recently, a mechanism-based MMP inhibitor, SB-3CT, was designedaccording to the X-ray crystallographic information of the MMP activesite (Brown et al., 2000). X-ray absorption studies revealed thatbinding of this molecule to the catalytic zinc reconstructs theconformational environment around the active site metal ion back to thatof the pro-enzyme [Kleifeld et al., 2001, J Biol. Chem. 276: 17125-31].

In the context of a combination therapy, combination therapy compoundsmay be administered by the same route of administration (e.g.intrapulmonary, oral, enteral, etc.) that the described compounds areadministered. In the alternative, the agents for use in combinationtherapy with the herein described agents may be administered by adifferent route of administration.

The agent which down-regulates ECM-associated polypeptides can beadministered immediately prior to (or after) the anti-pathogenic agent,on the same day as, one day before (or after), one week before (orafter), one month before (or after), or two months before (or after) theanti-pathogenic agent, and the like.

The agents which down-regulate ECM-associated polypeptides and theanti-pathogenic agent can be administered concomitantly, that is, wherethe administering for each of these agents can occur at time intervalsthat partially or fully overlap each other. The agents described hereincan be administered during time intervals that do not overlap eachother. For example, the first agent can be administered within the timeframe of t=0 to 1 hours, while the second agent can be administeredwithin the time frame of t=1 to 2 hours. Also, the first agent can beadministered within the time frame of t=0 to 1 hours, while the secondagent can be administered somewhere within the time frame of t=2-3hours, t=3-4 hours, t=4-5 hours, t=5-6 hours, t=6-7 hours, t=7-8 hours,t=8-9 hours, t=9-10 hours, and the like. Moreover, the second agent canbe administered somewhere in the time frame of t=minus 2-3 hours,t=minus 3-4 hours, t=minus 4-5 hours, t=5-6 minus hours, t=minus 6-7hours, t=minus 7-8 hours, t=minus 8-9 hours, t=minus 9-10 hours.

The agents of the present invention are typically provided in combinedamounts to treat the infection and/or to reduce symptoms or diseaseassociated with a secondary infection. This amount will evidently dependupon the particular agent selected for use, the nature and number of theother treatment modality, the condition(s) to be treated, preventedand/or palliated, the species, age, sex, weight, health and prognosis ofthe subject, the mode of administration, effectiveness of targeting,residence time, mode of clearance, type and severity of side effects ofthe agents and upon many other factors which will be evident to those ofskill in the art.

The present inventors have shown that administration of an antibodywhich binds to and down-regulates MMP-14 prevents complications of asecondary infection. More specifically, the present inventors showedthat administration of an MMP-14 antibody together with an antiviralagent reduced the symptoms in animals infected with the influenza virus(as the primary infection) and S. pneumoniae (as the secondaryinfection).

Thus, the present inventors propose that administration of agents whichspecifically down-regulate ECM-associated polypeptides and anantipathogenic agent may prevent (or reduce the symptoms of) a secondaryinfection.

Thus, according to another aspect of the present invention there isprovided a method of treating or preventing a disease associated with asecondary infection in a subject infected with a pathogen comprisingadministering to the subject a therapeutically effective amount of ananti-pathogenic agent directed towards the pathogen and atherapeutically effective amount of an agent which down-regulates anECM-associated polypeptide, thereby treating or preventing the diseaseassociated with the secondary infection in the subject.

As used herein, the phrase “secondary infection” refers to an infectionthat occurs during or after treatment of another pre-existing infection.It may result from the treatment itself or from changes in the immunesystem.

The term “preventing” refers to inhibiting or arresting the developmentof the secondary infection and/or causing the prevention, reduction,remission, or regression of symptoms of the secondary infection.

According to a particular embodiment, the combination therapy proposedby the present invention reduces the complications or treats a disease(e.g. sepsis) associated with the secondary infection.

The secondary infection may be a bacterial infection, a viral infectionor a fungal infection.

The primary and the secondary infections are typically infections of thesame organ (e.g. lungs and/or respiratory tract).

In one embodiment, the primary infection is viral infection (e.g.influenza) and the secondary infection is a bacterial infection (e.g. S.pneumoniae).

In another embodiment, the primary infection is a bacterial infectionand the secondary infection is a viral infection.

In yet another embodiment, the primary infection is viral infection andthe secondary infection is a fungal infection.

In yet another embodiment, the primary infection is a bacterialinfection and the secondary infection is a fungal infection.

According to yet another aspect of the present invention there isprovided a method of treating influenza in a subject in need thereofcomprising administering to the subject a therapeutically effectiveamount of an agent which down-regulates at least one ECM-associatedpolypeptide, thereby treating the influenza.

ECM-associated polypeptides have been described herein above. Accordingto a particular embodiment, the ECM-associated polypeptide is set forthin Table 1—for example MT1-MMP1.

According to a particular embodiment, treatment of influenza is effectedby administering an antibody which down-regulates an amount of MT1-MMP1,such as those described herein above.

In order to prevent the collapse of the ECM, preferably, the agent isprovided no more than 5 days after the start of symptoms of theinfluenza virus, no more than 4 days after the start of symptoms of theinfluenza virus, no more than 3 days after the start of symptoms of theinfluenza virus, no more than 2 days after the start of symptoms of theinfluenza virus, and even no more than 1 day after the start of symptomsof the influenza virus.

In any of the method and uses described herein, the agents can be usedper se or in a pharmaceutical composition which further comprises apharmaceutically (or cosmetically) acceptable carrier.

In one embodiment, the agents are co-formulated in the samepharmaceutical composition.

In another embodiment, the agents are formulated in separatepharmaceutical compositions. The separate pharmaceutical compositionsmay be comprised in a single article of manufacture, e.g. a kit.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to any of the agentsdescribed herein. It will be appreciated that the pharmaceuticalcompositions may comprise additional active agents known to be useful intreating a particular disease.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, intraperitoneal, intranasal, orintraocular injections.

According to a particular embodiment, the route of administration is viatopical delivery.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum Arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (e.g. the compounds described herein) effective toprevent, alleviate or ameliorate symptoms of a disorder (e.g., fibroticor inflammatory disease) or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated from animalmodels to achieve a desired concentration or titer. Such information canbe used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures inexperimental animals. The data obtained from these animal studies can beused in formulating a range of dosage for use in human. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provide cellnumbers sufficient to induce normoglycemia (minimal effectiveconcentration, MEC). The MEC will vary for each preparation, but can beestimated from in vitro data. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.Detection assays can be used to determine plasma concentrations.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

It is expected that during the life of a patent maturing from thisapplication many relevant antiviral/antibacterial agents will bedeveloped and the scope of the term antiviral/antibacterial is intendedto include all such new technologies a priori.

As used herein the term “about” refers to ±10%

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W.H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and Methods

Influenza Virus and Streptococcus pneumoniae Bacterial Agent:

Mouse-adapted PR8 virus, influenza A/Puerto Rico/8/34 (A/PR/8/34, H1N1)was persistently grown in hen egg amnion and influenza effective titerswere quantified as previously described (Achdout et al., 2003).Streptococcus pneumoniae (S. pneumoniae) D39 type 2 encapsulated strainwas grown in Todd-Hewitt broth (Difco Laboratories) For isolation andinfection of mice, bacteria were grown overnight on tryptic soy agar(Hylab Laboratories) supplemented with 3% (vol/vol) sheep erythrocyte at37° C. and were then harvested by centrifugation at 4000 g for 20 min topellet the bacteria and dilute it to the desired concentration.

Infection Procedures:

Female C57BL/6J mice (4-5 weeks of age) were anesthetized withketamine-xylazine and were intra-nasally inoculated with 50 μl ofdiluted virus. The same stock was used for all the experimentscontaining influenza A/Puerto Rico/8/34 (A/PR/8/34, H1N1) strain, 9×10⁷PFU/ml, HA 1:1,024. To study pathogenesis of Influenza infection,C57BL/6 mice were intra-nasally infected with 4×10³ PFU of influenza PR8virus equivalent to lethal dose. Mice were sacrificed on 3, 7, 11, 26,32, 49, 74, 98, 122, 148 hours post infection and the lungs wereharvested and homogenized for RNA isolation. To study the effectivity ofanti-MT1-MMP inhibitor, both in the single viral infection model and inthe double infection model combining S. pneumoniae, a sub-lethal dose of800 PFU was used, which was diluted accordingly and administered alongthe same route. S. pneumoniae was grown on tryptic soy agar (HylabLaboratories) supplemented with 3% (vol/vol) sheep erythrocytes. Thebacterium was diluted in sterile PBS and administered intra-nasally 4days post viral infection at a dose of 30 CFU, in a volume of 50 μl. Themice were anesthetized and held in an upright position while inoculated.Mice were weighted and monitored at least daily for illness andmortality. All animal procedures were performed according to IACUCguidelines and were approved by the committee of the Weizmann Instituteof Science.

Treatment of Animals:

Mice were treated in the single infection experiments as well as in theco-infection experiments with 3 mg/kg of LEM 2/15 Fab fragment at atotal volume of 100 μl per injection, given intra-peritoneally everyday. GST-Fab, designated in text as control Fab, served as non-relevantcontrol and was given the same dose as the LEM 2/15 treated group. PBSused as a vehicle control.

LEM 2/15 (Anti-MTI-MMP Ab) Purification:

Hybridoma cells of LEM-2/15 were grown in DCCM (serum-free mediumdesigned for hybridoma cell growth and monoclonal antibody production,purchased from Biological Industries). Cells were precipitated bycentrifugation at 193 g, and the supernatant was collected. Thesupernatant was dialyzed against 20 mM phosphate buffer (pH 8). A 1 mlHiTrap protein A high-performance column was equilibrated with 100 mMphosphate buffer (pH 8), and the supernatant was loaded at 1 ml/min. Theantibody was eluted with 100 mM citrate buffer (pH 6) and dialyzedagainst 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl.

Antibody Digestion with Papain:

Papain was activated in 0.5 M Tris-HCl (pH 8), 10 mM EDTA, and 5 mMdithiothreitol for 15 min at 370 C. Active papain was added to asolution of intact LEM-2/15 at a ratio of 1:1,000, and the digestionprocess was carried out for 3 h at 370 C. The digestion reaction wasterminated with the addition of 20 mM iodoacetamide in the dark at roomtemperature for 30 min. The Fab fragment was isolated from the Fc by aprotein A column, and the Fab fragment was collected from the flowthrough and dialyzed against 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl.The purity of the Fab fragment was estimated by 12% SDS-PAGE gel. PureFab fragment was filtered to assure sterility and kept at −80° C.conditions until use.

Glutathione S-transferases (GST)-Fab Fragment:

Fab fragment from the whole GST antibody were produced as described inthe upper section (Antibody Digestion with Papain).

Quantification of Viral and Bacterial Loads:

Viral titers in the lungs were determined by titration of organhomogenate on MDCK cells and plaque forming units (PFUs) were quantifiedas described in (Okuda et al., 2001). Strep. pneumoniae levels weredetermined by plating titrated amounts of organ homogenate on trypticsoy agar plates supplemented with 3% sheep erythrocytes (HylabLaboratories). Organs were homogenized using the GentleMACS 1 ml ofappropriate buffer for PFUs or 10 ml of sterile water for Strep.pneumoniae for CFUs. Viral burdens were also quantified using qPCR, asdescribed before for the detection of virus in patients (Hindiyeh etal., 2005). S. pneumoniae identification was done using qPCR aspreviously described (Ogunniyi et al., 2002). Serial dilutions ofInfluenza A (A/PR/8/34) virus titrated on Madin-Darby Canine Kidney(MDCK) cells were used as standards to determine the quantity of theinfluenza virus by quantitative real-time PCR (qRT-PCR) and convert theqPCR results into viral load numbers.

RNA Isolation:

Lungs were removed and immediately transferred into RNA Latter solution(Invitrogen). For RNA isolation, the lung was cut into small pieces inthe presence of QIAzol, homogenized using SPEX CertiPrep homogenizer,and total RNA was extracted with a miRNeasy Mini Kit (Qiagen). RNAintegrity was determined (Tapestation, Agilent Technologies) andconcentration measured with a Qubit Fluorometric Quantitation device(LifeTechnologies).

Preparation of RNA Sequencing Libraries:

For RNA-seq a derivation of MARS-seq technique was used as described in(Jaitin et al., 2014). In brief, total RNA was fragmented into fragmentshaving an average size of 300 nucleotides by chemical heat (95° C.)treatment for 4:30 min (NEBNext Magnesium RNA Fragmentation Module). The3′ polyadenylated fragments were enriched by selection on poly dT beads(Dynabeads Invitrogen). Strand-specific cDNA was synthesized using apoly T-VN oligo (18 T) and Affinity Script RT enzyme (Agilent).Double-strand DNA was obtained using Second strand synthesis kit (NEB).DNA ends were repaired using T4 polynucleotide kinase and T4 polymerase(NEB-Next). After the addition of an adenine base residue to the 5′ endusing Klenow enzyme (NEB-Next), a barcode Illumina compatible adaptor(IDT) was ligated to each fragment. The washed DNA fragment wasamplified by PCR (12 cycles) using specific primers (IDT) to the ligatedadaptors. The quality of each library was analyzed by TapeStation(Agilent).

Pre Processing of RNASeq Data:

RNA-seq was performed as described in Lavin et al., 2014. In brief, allreads, both from whole lung (FIGS. 1A-1D) and cell populations (FIG. 8)were aligned to the mouse reference genome (NCBI 37, MM9) using theTopHat aligner. Normalized expression table was created using ESATgarberlabdotumassmeddotedu/software/esat/based on the negative binomialdistribution and a local regression model. Data manipulation-Discardgenes from table that have values>0 only once; Calculate 75 percentileof data result is 33 (set noise to 32); Find max of raw and discard ifmax<32; log 2 values as x+32; Average replicates; keep rows wheremax−min>0.8 (notice not 2 fold but 1.75); K-Means in matlab for 20clusters; manually ordered the clusters for visual purpose picture wasdone in GeneE.

qPCR:

Total RNA was reverse transcribed to cDNA using high capacity cDNAreverse transcription kit (Applied Biosystems). RT-PCR was performedwith LightCycler480 SYBR green I master mix (Roche) in triplicate, usingGAPDH and β-actin for normalization. Primer list is provided in Table2A, herein below.

TABLE 2A Gene Direction sequence SEQ ID MT1-MMP Forward5-AGCACTGGGTGTTTGACG-3 28 MT1-MMP Reverse 5-GTCTTCCCATTGGGCATC-3 29MMP-9 Forward 5-CAGACGTGGGTCGATTCC-3 30 MMP-9 Reverse5-TCATCGATCATGTCTCGC-3 31 MMP-8 Forward 5-GCAGCGCTTCTTCAGCTT-3 32 MMP-8Reverse 5-GTGTGTGTCCACTTGGGA-3 33 MMP-2 Forward 5-ACGATGATGACCGGAAGT-334 MMP-2 Reverse 5-GTGTAGATCGGGGCCATC-3 35 TIMP1- Forward5-GCAGTGATTTCCCCGCCA-3 36 var2 TIMP1- Reverse 5-GGGGGCCATCATGGTATC-3 37var2 MMP-3 Forward 5-AAGGAGGCAGCAGAGAAC-3 38 MMP-3 Reverse5-GCACTGTCATGCAATGGG-3 39 LGMN Forward 5-GCCTACCAGATCATCCAC-3 40 LGMNReverse 5-ACATCTGTGCCGTTAGGT-3 41 GZMB Forward 5-ACAACACTCTTGACGCTG-3 42GZMB Reverse 5-CGAGAGTGGGGCTTGACT-3 43 LCN2 Forward5-ACAACCAGTTCGCCATGG-3 44 LCN2 Reverse 5-AAGCGGGGTGAAACGTTCC-3 45 PR8Forward 5-GACCRATCCTGTCACTGAC-3 46 MATRIX A INF A-CDC PR8 Reverse5-TGCAGTCCTCGCTCACTGG 47 MATRIX GCACG-3 A INF A-CDC 16S rRNA Forward5-GGTGAGTAACGCGTAGGTAA-3 48 16S Rrna Reverse 5-ACGATCCGAAAACCTTCTTC-3 49TIMP-2 Forward TCTAGGAGTCCCAGTCAGCC 50 TIMP-2 ReverseCAACAAGGACTGCCAAGCAC 51 GAPDH Forward GCCCTTGAGCTAGGACTGGA 52 GAPDHReverse TACGGCCAAATCCGTTCACA 53

In Gel Proteolysis and Mass Spectrometry Analysis:

Lung samples were de-cellularized using 0.5% EDTA supplemented with 2%triton, shaking for 24 hours. Samples were then dehydrated using aSpeedvac and weighted. Samples were then subjected to in-solutiondigestion using activated MMP-13, 500 nM in TNC buffer in (50 mMTris-HCl, 150 mM NaCl, 5 mM MgCl2, 5 mM CaCl2, pH 7.4) a volume of120μl, for 24 hours shaking at 30° C. The volume and concentration ofactivated MMP-13 were adjusted according to the weight of each sample,and each sample was run in duplicates. Protein extract was loaded onSDS-PAGE for a short run. The proteins in the gel were reduced with 2.8mM DTT (60° C. for 30 min), modified with 8.8 mM iodoacetamide in 100 mMammonium bicarbonate (in the dark, room temperature for 30 min) anddigested in 10% acetonitrile and 10 mM ammonium bicarbonate withmodified tryp sin (Promega) at a 1:10 enzyme-to-substrate ratio,overnight at 37° C. An additional second trypsinization was done for 4hours. The resulting tryptic peptides were resolved by reverse-phasechromatography on 0.075×200-mm fused silica capillaries (J&W) packedwith Reprosil reversed phase material (Dr Maisch GmbH, Germany). Thepeptides were eluted with linear 95 minutes gradients of 7 to 40% and 8minutes at 95% acetonitrile with 0.1% formic acid in water at flow ratesof 0.25 μl/min. Mass spectrometry was performed by an ion-trap massspectrometer (Orbitrap XP, Thermo) in a positive mode using repetitivelyfull MS scan followed by collision induces dissociation (CID) of the 7most dominant ion selected from the first MS scan.

The mass spectrometry data was analyzed using the MaxQuant 1.3.0.5software searching against the mouse section of the Uniprot databasewith mass tolerance of 20 ppm for the precursor masses. Peptide- andprotein-level false discovery rates (FDRs) were filtered to 1% using thetarget-decoy strategy. Protein table were filtered to eliminate theidentifications from the reverse database, and common contaminants andsingle peptide identifications. The data was quantified by label freeanalysis using the same software, based on extracted ion currents (XICs)of peptides enabling quantitation from each LC/MS run for each peptideidentified in any of experiments. To search for ECM related proteins,annotations were determined using the GORILLA Bioinformatics Resources.

Western Blot:

Lung samples were homogenized using gentleMACS (Miltenyi Biotec)according to manufacturer instructions using 500μl rippa buffercontaining protease inhibitor cocktail (Roche). Protein levels were thenmeasured using BCA kit (Pierce Biotechnology) and run in duplicates onSDS-PAGE gel using a mini-electrophoresis apparatus (Bio-RadLaboratories, Inc.). The resolved polypeptides were transferred onto anitrocellulose membrane in Tris-glycine buffer containing 25% methanol.The membranes were blocked with 5% dried milk, and then incubated withgoat anti-MMP-8 (SantaCruz), rabbit anti-MMP-9 (Abcam) or rabbitanti-MT1MMP (Abcam) antibodies. Rabbit anti-GAPDH (SantaCruz) wasincluded in each procedure to avoid inter-assay variations.Nitrocellulose membranes were incubated with goat anti-rabbit HRPconjugated antibody (Abcam), or bovine anti-goat HRP (Sigma). Membraneswere developed using EZ-ECL chemiluminescence detection kit (Biologicalindustries). A molecular mass protein standard (PageRuler PrestainedProtein ladder, Fermentas) was included in each assay.

Lung Preparation for Imaging:

Lungs were inflated by using PBS for in situ zymography or 4% PFA forother imaging purposes. This was done by exposing the trachea, insertinga cannula 22G, 0.8×25 mm, (Cathy IV cannula, HMD Healthcare LTD) andinjecting 5 ml fluid. The cannula was ligated to the trachea to avoidspillage. Mouse lungs were harvested at different time points postinfection, embedded in OCT and frozen in −80° C. until analyzed.

Two-Photon Microscopy and Second Harmonics Generation:

Before imaging, lungs were cut 300 μm and immediately visualized using atwo-photon microscope in the in-vivo imaging unit in the Weizmanninstitute (2 PM:Zeiss LSM 510 META NLO; equipped with a broadband MaiTai-HP-femtosecond single box tunable Ti-sapphire oscillator, withautomated broadband wavelength tuning 700-1,020 nm from Spectraphysics,for two-photon excitation). For collagen second harmonic imaging awavelength of 800 nm was used (detection at 400 nm).

AirSEM and SEM Imaging of Intact Lung Tissues and Lung ECM Scaffolds:

Fixed lungs were sectioned into 300 μm sections. Sections were washedthree times in a large volume of PBS to remove OCT remnants, followed bythree DDW washes. For tissue imaging, the slices were gently placed on aSuperFrost Plus glass slides and stained as previously described.Briefly, sections were washed with DDW and stained with 0.1% rutheniumred (EM grade, Sigma-Aldrich) in a 0.1 M sodium cacodylate buffer pH 7.4(analytical standard, Sigma-Aldrich) for 15 min. The sections were thenthoroughly washed with DDW and stained with a 2% uranyl acetate solutionfor 10 min. The samples were then washed with DDW and allowed to dry inthe air at room temperature for 5-7 min before airSEM™. ECM scaffoldswere first de-cellularized using 0.5% EDTA supplemented with 2% tritonfor 24 hours. Staining was done as previously described. Forconventional scanning electron microscopy (SEM) samples were furtherdehydrated through an ethanol series increasing in concentration to 100%ethanol, were dried in a critical point dryer and coated by Au/Palladiumaccording to standard sample preparation procedure for SEM imaging withan Ultra 55 Feg Zeiss SEM operating at 2 kV.

Immunohistochemistry:

Immunohistochemistry was performed using standard techniques on 10 μmcryo-sections. Sections were fixed with 4% PFA, blocked with 3% BSA,incubated overnight with primary rabbit anti-laminin (Sigma), or rabbitanti-lumican (abcam), or rabbit anti-collagen IV or rat antibody forF4/80 and CD45 cell surface protein (abcam). LEM2/15 was conjugated toAlexa Fluor 555 Protein by Labeling Kit (Molecular Probes), according tomanufacturer's instructions. Sections were then washed with PBS,incubated with a goat anti-rabbit HRP conjugated (Jackson). Fluoresceinor Cy3 conjugated anti-HRP kit was used (Perkin Ehlmer) respectively,followed by DAPI staining (Sigma) and mounting with immune-mount (ThermoScientific). Samples were imaged using Nikon 80i eclipse microscope.

Collagen Type I in Situ Zymography:

Non-fixed lungs samples were cut into 10 μm sections, gently washed toremove OCT. After washing a 1 mg/ml DQ collagen type I (MolecularProbes) was diluted to 40 mg/ml with developing buffer (50 mM Tris (pH7.5), 100 mM NaCl, 5 mM CaCl₂). Samples were incubated for 4 hours at37° C. the reaction was stopped with 4% paraformaldehyde and followed bythe desired immune-staining, mounted with immune-mount (ThermoScientific) and imaged using Nikon 80i eclipse microscope.

Relative Frequency of Fiber Orientation Analysis:

Imaging analysis was done by Fiji package, Directionality analysis.Graphs were plotted using GraphPad Prism 6. The relative frequency offiber spatial orientation was measured using the “Directionality” pluginanalysis tool in Fiji package version 6.1.1.

Flow Cytometry:

Lungs from infected and control uninfected C57BL/6J mice were immersedin cold PBS, cut into small pieces in 5 ml DMEM containing 10% bovinefetal serum (FACS buffer). Cell suspensions were grinded using 1-mlsyringe cup on a 70-lm cell strainers (BD Falcon). Cells were washedwith ice-cold PBS. Remaining red blood cells were lysed using ammoniumchloride solution (Sigma). Cells were harvested and immersed 1 ml FACSbuffer [PBS+2% FCS, 1 mM EDTA]. Lung cells were stained with antibodiesagainst multiple surface antigens: PE-conjugated LEM 2/15 (anti-mouseMT1-MMP ab), PerCP/cy5.5-conjugated anti-mouse CD45 (clone—F11) orPacific blue-anti-mouse CD45, APC-Cy7-conjugated EPCAM, APC-conjugatedanti-CD11b, PerCP/cy5.5-anti-mouse Ly6C, FITC-anti-mouse Ly6G (clone1A8), FITC-anti-mouse NKp46, FITC-anti-mouse-TCR-β. Flow cytometry wasperformed using FACSAriaIII Flow Cytometer (BD biosciences), and datawas analyzed using FlowjoV 10.0.8 software. Sorted cells fromnon-infected control and infected mice treated with PBS were furthersubjected to RNA extraction, as previously mentioned in RNA extractionsection, and were sequenced using RNA-Seq profiling and qPCR.

Example 1 Extra Cellular Matrix Genes are Induced During InfluenzaInfection

In order to systematically describe the effect of influenza inflectionon host ECM circuits, genome-wide RNA-seq was used to measure thetemporal transcriptional response of whole lung tissue during aseven-day course of influenza (Altbourn et al., 2014). Gene expressionwas measured at ten time points following infection. C57BL/6 mice wereinfected by intranasal inoculation of mouse-adapted PR8 influenza A H1N1virus using either lethal or sub-lethal dosages. PR8 infection is widelyused as a influenza infection model (Morens et al., 2008; Tate et al.,2011; Taubenberger and Morens, 2006; Watanabe et al., 2013) consistingof rigorous alveolar spread, acute pulmonary hemorrhage and intensivehost responses. Along with the disease progression, the symptoms andloss of body weight are initiated 24-48 hours post infection with anincrease in viral load in the lungs (FIG. 6D). As expected, influenzainfection resulted in induction of genes that are involved ininflammation chemotaxis (CXCL1, CXCL10, II1b, II1r), defense againstviral infection (ISG15, IFNB1, IRF7, IFIT1 and IFIT3) and variouschemokines (CCL2, CCL3, CCL4, CXCL2) as well as down-regulation of genesrelated to lung homeostasis (secretoglobins and relevant transcriptionfactors (e.g. NK×2.1)) (FIG. 1A). Furthermore, many genes which weredown-regulated following infection belong to oxygen-reduction processes,surfactant homeostasis (SFTPA1, SFTPC), cell-cell adhesion moleculessuch as integrins, cadherin, claudin (CLDN18, ITGB2, CDH5) and lipidmetabolism (APOE, APOC1, APOA1BP). Additional genes that werestatistically significantly up-regulated are set forth in Table 2Bherein below.

TABLE 2B Symbols Genes (mouse) 1190002H23RIK NM_025427 1500012F01RIKNM_001081005 2010001M09RIK NM_027222 AA467197 NM_001004174 Acta1NM_009606 Actb NM_007393 Adam15 NM_001037722 Adam8 NM_007403 Adamts15NM_001024139 Adamts4 NM_172845 Aif1 NM_019467 Alpl NM_007431 Angptl4NM_020581 Apod NM_007470 Apol6 NM_028010 Apol9a NM_001162883 Apol9bNM_173743 Arrb2 NM_145429 Atf3 NM_007498 AW112010 NM_001177351 B2mNM_009735 B4galt1 NM_022305 Bak1 NM_007523 Batf2 NM_028967 Bcl3NM_033601 Bdkrb1 NM_007539 Bgn NM_007542 Bst2 NM_198095 C1qa NM_007572C1qb NM_009777 C1qc NM_007574 C1qtnf6 NM_028331 C3ar1 NM_009779 Casp4NM_007609 Ccl2 NM_011333 Ccl20 NM_001159738 Ccl4 NM_013652 Ccl5NM_013653 Ccl7 NM_013654 Cd274 NM_021893 Cd300lf NM_001169153 Cd3dNM_013487 Cd72 NM_001110322 Cd8a NM_009857 Cd8b1 NM_009858 Cdca3NM_013538 Cdkn1a NM_007669 Cebpd NM_007679 Cfb NM_001142706 CideaNM_007702 Ckap4 NM_175451 Ckm NM_007710 Cldn4 NM_009903 Cmklr1 NM_008153Cmpk2 NM_020557 Col1a1 NM_007742 Col1a2 NM_007743 Col3a1 NM_009930Cox7a1 NM_009944 Cpxm1 NM_019696 Csrnp1 NM_153287 Ctgf NM_010217 CtpsNM_016748 Ctss NM_021281 Ctsz NM_022325 Cxcl1 NM_008176 Cxcl10 NM_021274Cxcl12 NM_021704 Cxcl13 NM_018866 Cxcl16 NM_023158 Cxcl2 NM_009140 Cxcl5NM_009141 Cxcl9 NM_008599 Cyp4f18 NM_024444 Cyr61 NM_010516 DaxxNM_001199733 Dbp NM_016974 Ddit4 NM_029083 Ddx58 NM_172689 Dhx58NM_030150 Dntt NM_009345 Dtx31 NM_001013371 Ecm1 NM_007899 Edem1NM_138677 Eif2ak2 NM_011163 Eln NM_007925 Epha2 NM_010139 Epsti1NM_029495 F3 NM_010171 Fam26f NM_175449 Fbn1 NM_007993 Fcer1g NM_010185Fcgr1 NM_010186 Fcgr4 NM_144559 Fgfr1 NM_001079908 Fkbp5 NM_010220 FlnbNM_134080 Fn1 NM_010233 Fscn1 NM_007984 Fst NM_008046 Fxyd5 NM_001111073Gadd45g NM_011817 Gbp10 NM_001039646 Gbp2 NM_010260 Gbp3 NM_018734 Gbp4NM_008620 Gbp5 NM_153564 Gbp6 NM_194336 Gbp9 NM_172777 Glycam1 NM_008134Gm12250 NM_001135115 Gm13889 NM_001145034 Gm14446 NM_001101605 Gm4841NM_001034859 Gm4951 NM_001033767 Gpd1 NM_010271 Gpx3 NM_008161 GrnNM_008175 Gvin1 NM_001039160 Gzmb NM_013542 H2-Q7 NM_010394 H2-Q9NM_001201460 H2-T10 NM_010395 H2-T22 NM_010397 H2-T23 NM_010398 H2-T9NM_010399 Has1 NM_008215 Hcls1 NM_008225 Helz2 NM_183162 Hmga1NM_001166537 Hmga1-rs1 NM_001166477 Hspa8 NM_031165 I830012O16RikNM_001005858 Ier5 NM_010500 Ifi203 NM_001045481 Ifi204 NM_008329 Ifi205NM_172648 Ifi2712a NM_029803 Ifi35 NM_027320 Ifi44 NM_133871 Ifi47NM_008330 Ifih1 NM_027835 Ifit1 NM_008331 Ifit2 NM_008332 Ifit3NM_010501 Ifitm3 NM_025378 Igtp NM_018738 Iigp1 NM_001146275 Il10raNM_008348 Il18bp NM_010531 Il1b NM_008361 Il1rn NM_001039701 Il21rNM_021887 Irf1 NM_001159396 Irf5 NM_012057 Irf7 NM_016850 Irf8 NM_008320Irg1 NM_008392 Irgm1 NM_008326 Irgm2 NM_019440 Isg15 NM_015783 Isg20NM_020583 Itga5 NM_010577 Junb NM_008416 Kcnn4 NM_001163510 Krt13NM_010662 Krt4 NM_008475 Laptm5 NM_010686 Lars2 NM_153168 LckNM_001162433 Lcn2 NM_008491 Lgals3bp NM_011150 Lgals9 NM_001159301 LgmnNM_011175 Lilrb4 NM_013532 Lox NM_010728 Loxl1 NM_010729 Loxl2 NM_033325Loxl3 NM_013586 Ly6a NM_010738 Ly6c1 NM_010741 Ly6c2 NM_001099217 Ly6iNM_020498 Lyve1 NM_053247 Mmp14 NM_008608 Mmp3 NM_010809 Mmp8 NM_008611Mnda NM_001033450 Mndal NM_001170853 Mpeg1 NM_010821 Ms4a4b NM_021718Ms4a4c NM_029499 Ms4a6b NM_027209 Ms4a6c NM_028595 Ms4a6d NM_026835 Mt1NM_013602 Mt2 NM_008630 Mx1 NM_010846 Mx2 NR_003508 Mxd1 NM_010751 Myh1NM_030679 Myh8 NM_177369 Mylpf NM_016754 Nampt NM_021524 NfkbiaNM_010907 Nlrc5 NM_001033207 Nppa NM_008725 Nt5c3 NM_026004 Oas1aNM_145211 Oas1g NM_011852 Oas2 NM_145227 Oasl1 NM_145209 Oasl2 NM_011854Ogfr NM_031373 Parp12 NM_172893 Parp14 NM_001039530 Parp9 NM_030253 Per1NM_001159367 Pfkfb3 NM_001177757 Phf11b NM_001164327 Phf11d NM_199015Pirb NM_011095 Pla2g7 NM_013737 Plac8 NM_139198 Plat NM_008872 PlauNM_008873 Pld4 NM_178911 Pnp NM_013632 Pnp2 NM_001123371 Pou2af1NM_011136 Ppa1 NM_026438 Prmt1 NM_019830 Prss22 NM_133731 Psmb10NM_013640 Psmb8 NM_010724 Psme2 NM_011190 Pstpip1 NM_011193 PtafrNM_001081211 Ptk7 NM_175168 Ptx3 NM_008987 Pycard NM_023258 Pyhin1NM_175026 Qsox1 NM_001024945 Relb NM_009046 Retnla NM_020509 Rhox8NM_001004193 Rpsa NM_011029 Rsad2 NM_021384 Rtp4 NM_023386 S100a14NM_001163526 S100a4 NM_011311 S100a6 NM_011313 S100a8 NM_013650 S100a9NM_009114 Saa1 NM_009117 Saa3 NM_011315 Samd9l NM_010156 Samhd1NM_001139520 Sbno2 NM_183426 Sdc3 NM_011520 Sell NM_001164059 Sema7aNM_011352 Serinc3 NM_012032 Serpina3f NM_001033335 Serpina3g NM_009251Serpina3m NM_009253 Serpina3n NM_009252 Serpine1 NM_008871 Serping1NM_009776 Sfn NM_018754 Sh3pxd2b NM_177364 Slc15a3 NM_023044 Slc25a37NM_026331 Slc7a5 NM_011404 Slfn1 NM_011407 Slfn2 NM_011408 Slfn4NM_011410 Slfn5 NM_183201 Slfn8 NM_001167743 Slfn9 NM_172796 Snx32NM_001024560 Socs1 NM_009896 Socs3 NM_007707 Sparcl1 NM_010097 Sphk1NM_011451 Spp1 NM_009263 Sprr1a NM_009264 Stat1 NM_009283 Stat2NM_019963 Tap1 NM_013683 Tap2 NM_011530 Tapbp NM_001025313 Tcf7NM_009331 Tgfbi NM_009369 Tgm2 NM_009373 Tgtp1 NM_011579 Tgtp2NM_001145164 Thbs1 NM_011580 Themis2 NM_001033308 Thrsp NM_009381 Thy1NM_009382 Timp1 NM_001044384 Tinagl1 NM_001168333 Tnc NM_011607 Tnfaip2NM_009396 Tnfrsf12a NM_013749 Tnni2 NM_009405 Tor3a NM_023141 Tpm2NM_009416 Trafd1 NM_172275 Trex1 NM_011637 Trib1 NM_144549 Trim25NM_009546 Trim30a NM_009099 Tuba1c NM_009448 Tubb5 NM_011655 Tubb6NM_026473 Ubc NM_019639 Ucp1 NM_009463 Usp18 NM_011909 Vcan NM_001134475Wars NM_001164488 Xaf1 NM_001037713 Xdh NM_011723 Zbp1 NM_021394 Znfx1NM_001033196

Of special notice was a large group of genes involved in ECM remodelingincluding macromolecule metabolism and protease synthesis (FIG. 1A).Remarkably, this group of genes was highly over-represented throughoutinfection, exhibiting a wide panel of pathways involved in multiple ECMremodeling events (FIG. 1A-1C). An enrichment of functional categoriesrelating to extra cellular modulators involved in proteolysis, collagenremodeling and catabolism, fibrinolysis, wound healing, homeostasis andcell migration (p≤10⁴) was uncovered, peaking 74 hours post infection(FIG. 1B). All together, 479 out of 3530 differentially expressed genes(13.6%) are related to ECM remodeling. These included serine proteases,lysyl oxidases, cathepsins, disintegrins (ADAMs), metalloproteinases(MMPs) and their natural inhibitors (TIMPs), which serve as ECMmodifiers that determine the turnover of different ECM components.Within this group of genes, robust induction of MT1-MMP at both the RNA(400 fold change; FIG. 1D) and protein levels 48 hours post infection(FIG. 6A-B) was found. Using quantitative real time PCR, the temporalchanges in MT1-MMP expression was corroborated as well as otherrepresentative genes belonging to the MMP family (MMP-3, 8 and 9) andmodulating the ECM (FIG. 6A, 6B, 6C).

Example 2 MTI-MMP Expression is Induced in Myeloid Cells Post InfluenzaInfection

In order to identify the cell population acting as the source of MT1-MMPduring infection course by flow cytometry was performed. Of thepopulation of cells expressing MT1-MMP in the non-infected lung, theoverwhelming majority was non-hematopoietic cell population (CD45⁻,89.5%), while only 10.5% were of hematopoietic origin (CD45⁺) (FIG. 2A).Post infection, the CD45+MT1-MMP expressing population increasedfour-fold (40.9%). Specifically, the CD11b⁺.MT1-MMP⁺ portion of theimmune cells increased from 32.9% to 64.9%, while the non-hematopoietic(CD45⁻) MT1-MMP-expressing cells decreased by two-fold (FIG. 2A).Histogram plots (FIG. 2B) further show that the overall increase inMT1-MMP expression post infection (FIG. 2B) can be associated withincreased expression of MT1-MMP in CD11b+ cells (FIG. 2B), rather thanlung epithelial cells, in which MT1-MMP was reduced post infection (FIG.2B). MT1-MMP expression in CD45⁺ versus CD45⁻ sorted populations beforeand during infection was further validated at the RNA level using qPCR(FIG. 2C). Immunostaining for MT1-MMP as well as F4/80 markers ininfluenza-infected lungs confirm our observation that MT1-MMP expressingcells largely co-localize with F4/80 positive cells at 74 hours postinfection. Since macrophages are both CD11b+ and F4/80+ immune cells,these findings suggest that macrophages are a significant source ofMT1-MMP following infection (FIG. 7A arrow heads, 7B). In order tomonitor the collagenase activity of influenza-infected lungs, in situzymography was used. It was found that following infection,collagenolytic activity is mostly associated with CD45+ cells, as wellas CD45⁻ cells lining the bronchi of infected lungs (FIG. 7C arrows, D).

In order to further characterize the MT1-MMP-expressing populationsbefore and after (74 hours) infection, RNA-seq analysis was performed onsorted MT1-MMP-expressing CD45+ and CD45⁻ subpopulations. In total, 2169genes were found to be differentially expressed in cells post-infectionas compared to un-infected cells in both CD45+ and CD45⁻ populations(FIG. 8). Consistent with the analysis from whole lung RNA-seq, anincrease in activation of inflammatory signaling pathways and cytokineproduction in both immune (CD45+) and stromal (CD45⁻) cells expressingMT1-MMP following infection was observed (FIG. 8). The immune cells, inparticular, exhibited significant up-regulation of cytokines (CCL2,CCL3, CCL4, CXCL2, IL1b) and anti-viral response genes (e.g. SLFN4,IFIT1 and IFIT2), while the stromal cells exhibited down-regulation ofgenes associated with lung homeostatic functions such as surfactantproduction (e.g. SFTPB, SFTPC). The immune population showed increase inexpression of monocyte/macrophage/DC markers (e.g. CD11b) and decreaseof multiple B cell markers (e.g. CD19, CD37, CD79). Thus, MT1-MMPexpression post-infection is associated with activated immune cells fromthe myeloid compartment (FIG. 8).

Example 3 Influenza Infection Induces Destruction of ECM Morphology andComposition

MT1-MMP plays a major role in cancer-associated invasion processesthrough degradation of fibrillar collagen, laminin and other ECMcomponents. To evaluate the functional role of MT1-MMP in influenzainfection, mass spectrometry analysis (FIG. 3A) as well as scanningelectron microscopy (SEM) imaging of lung tissues devoid of its cellularcompartment (de-cellularized) before and after infection was performed(FIGS. 9A-9B). SEM analysis of influenza-infected lungs showed massivedistortion of ECM morphology (FIGS. 3B-3C) as well as rearrangement ofcollagen fibers, specifically in the alveolar walls. At 74 hourspost-infection, collagen bundles on the boundaries of alveolar sacsdisplayed unraveled fiber ends and dispersed orientation angles (FIG.3B). This was further confirmed by measuring the orientation of thefibrils composing the alveolar walls (FIG. 3C). In addition, thealveolar space and septa were distorted in the infected lungs (FIG. 3B).To validate these results and further analyze the integrity of the wholetissue—including the cells and ECM in their native environment—duringinfection, a novel form of electron microscopy imaging, AirSEM(Solomonov et al., 2014) was used. AirSEM enables visualization ofnative hydrated tissues in ambient conditions, thus avoiding potentialartifacts associated with sample preparation for SEM. Imaging ofvirally-infected lung tissues exhibited tissue destruction characterizedby both alveolar and bronchial cell depletion as well as distortion ofalveolar sacs and ducts followed by alveolar wall thinning (FIGS.9A-9B). Finally, AirSEM imaging of fresh lung ECM scaffolds(de-cellularized tissues) showed similar alveolar collagen degradationand distortion patterns as observed in conventional SEM analysis (FIGS.9A-9B).

Example 4 Global Proteomics Analysis Identifies Degradation of ECMScaffolds During Influenza Infection

In order to globally examine the proteolytic implications on ECMremodeling during influenza infection, a tandem mass spectrometry(LC-MS-MS) approach was used (FIG. 3A, Table 3, herein below). Analysisof the de-cellularized lung tissue 74 and 120 hours post infectioncompared with non-infected tissue showed compositional changes thatcould be connected to structural changes observed in the architecture ofcollagen fibrils lining the alveolar wall (FIGS. 3B-3C). Specifically,the proteomic data analysis of the influenza-infected lungs identifiedmodifications in the molecular composition of the ECM, including gradualdepletion of collagen and subtypes of laminin molecules (FIG. 3A). Inaddition, multiple basement-membrane-associated components as well asbasal-cell-adhesion molecules (e.g. Nidogen, Decorin, Collagen types IV,XII, XIV and Fibrillin; FIG. 3A) were depleted from infected lung tissueindicating massive transformation of ECM integrity and molecularcomposition. The loss of representative ECM molecules (Collagen IV,Decorin and Lumican) was confirmed by staining the ECM scaffolds ofinfected and non-infected lungs (FIGS. 3D-3I). Importantly, several ofthe components depleted during infection, such as type I collagen,laminin nidogen, lumican, mimecan, fibrillin and decorin, are knownMT1-MMP-substrates (Koziol et al., 2012; McQuibban et al., 2000; Noel etal., 2012; Overall, 2002; Shimizu-Hirota et al., 2012; Stegemann et al.,2013). Since multiple MT1-MMP substrates are degraded during the courseof influenza infection, it was hypothesized that lung ECM proteolysisand host mortality can be protected by specifically blocking thecollagenase activity of MT1-MMP.

TABLE 3 Normalized Normalized Normalized Protein annotation control T72T120 ADP/ATP translocase 1 OS = Mus musculus GN = Slc25a4 6.1535759475.977276302 0 PE = 1 SV = 4 - [ADT1_MOUSE] ADP/ATP translocase 2 OS =Mus musculus GN = Slc25a5 6.223198168 5.887684499 0 PE = 1 SV = 3 -[ADT2_MOUSE] Advanced glycosylation end product-specific receptor6.473725601 0 0 OS = Mus musculus GN = Ager PE = 2 SV = 1 -[C5H3H4_MOUSE] Agrin OS = Mus musculus GN = Agrn PE = 4 SV = 1 -6.308175158 6.593093331 6.511599322 [M0QWP1_MOUSE] Alpha-amylase 1 OS =Mus musculus GN = Amy1 PE = 1 SV = 2 - 0 5.65986227 7.460711241[AMY1_MOUSE] Annexin A1 OS = Mus musculus GN = Anxa1 PE = 1 SV = 2 - 05.824702042 0 [ANXA1_MOUSE] Annexin A2 (Fragment) OS = Mus musculus GN =Anxa2 5.679465046 5.957616046 6.195405289 PE = 2 SV = 1 - [B0V2N8_MOUSE]Aquaporin-5 OS = Mus musculus GN = Aqp5 PE = 2 SV = 1 - 6.1627162995.961971735 0 [AQP5_MOUSE] Basal cell adhesion molecule OS = Musmusculus GN = Bcam 6.227912096 5.999952109 0 PE = 2 SV = 1 -[BCAM_MOUSE] Basement membrane-specific heparan sulfate proteoglycan7.415377503 7.322287706 7.230770907 core protein OS = Mus musculus GN =Hspg2 PE = 2 SV = 1 - [E9PZ16_MOUSE] Beta-actin-like protein 2 OS = Musmusculus GN = Actbl2 6.386138757 6.785714287 7.052084571 PE = 1 SV = 1 -[ACTBL_MOUSE] Beta-globin OS = Mus musculus GN = Hbb-b1 PE = 2 SV = 1 -6.447007246 6.964298502 6.938126292 [A8DUK4_MOUSE] Carboxylesterase 1DOS = Mus musculus GN = Ces1d PE = 1 5.527869285 5.634653635 0 SV = 1 -[CES1D_MOUSE] Caveolin (Fragment) OS = Mus musculus GN = Cav1 PE = 26.495955194 6.212414678 0 SV = 2 - [D3Z148_MOUSE] Chitinase-3-likeprotein 4 OS = Mus musculus GN = Chi3l4 5.78250122 0 0 PE = 1 SV = 2 -[CH3L4_MOUSE] Collagen alpha-1(I) chain OS = Mus musculus GN = Col1a17.075805627 7.028473097 6.753210524 PE = 1 SV = 4 - [CO1A1_MOUSE]Collagen alpha-1(III) chain OS = Mus musculus GN = Col3a1 6.9342075357.060366511 7.23163175 PE = 2 SV = 4 - [CO3A1_MOUSE] Collagenalpha-1(IV) chain OS = Mus musculus GN = Col4a1 7.695020525 7.6945675897.536290851 PE = 2 SV = 4 - [CO4A1_MOUSE] Collagen alpha-1(VI) chain OS= Mus musculus GN = Col6a1 6.86809465 6.551279889 6.15880609 PE = 2 SV =1 - [CO6A1_MOUSE] Collagen alpha-1(XII) chain OS = Mus musculus5.714611383 0 0 GN = Col12a1 PE = 4 SV = 1 - [J3KMS9_MOUSE] Collagenalpha-1(XIV) chain OS = Mus musculus 6.125016434 0 0 GN = Col14a1 PE = 2SV = 1 - [B7ZNH7_MOUSE] Collagen alpha-2(I) chain OS = Mus musculus GN =Col1a2 6.543280973 6.813995104 7.023591569 PE = 2 SV = 2 - [CO1A2_MOUSE]Collagen alpha-2(IV) chain OS = Mus musculus GN = Col4a2 7.5508276687.530963433 7.33268 PE = 2 SV = 4 - [CO4A2_MOUSE] 6213 Collagenalpha-2(VI) chain OS = Mus musculus GN = Col6a2 6.947369979 6.7924655626.635688357 PE = 2 SV = 3 - [CO6A2_MOUSE] Collagen alpha-3(IV) chain OS= Mus musculus GN = Col4a3 7.21593568 7.650127224 7.416342179 PE = 1 SV= 2 - [CO4A3_MOUSE] Collagenase 3 OS = Mus musculus GN = Mmp13 PE = 1 SV= 1 - 7.72568074 7.15902446 7.286304725 [MMP13_MOUSE] Decorin OS = Musmusculus GN = DSP PE = 2 SV = 1 - 6.22520272 0 0 [PGS2_MOUSE]Desmoglein-1-alpha OS = Mus musculus GN = Dsg1a PE = 2 5.5487470855.852724683 6.62085048 SV = 2 - [DSG1A_MOUSE] Desmoplakin OS = Musmusculus GN = Dsp PE = 2 SV = 1 - 6.162304582 6.382926331 6.992943381[DESP_MOUSE] Dimethylaniline monooxygenase [N-oxide-forming] 25.714661158 5.311917766 0 OS = Mus musculus GN = Fmo2 PE = 1 SV = 3 -[FMO2_MOUSE] Elongation factor 1-alpha 1 OS = Mus musculus GN = Eef1a15.598955869 5.764182366 0 PE = 1 SV = 3 - [EF1A1_MOUSE] EMILIN-1 OS =Mus musculus GN = Emilin1 PE = 1 SV = 1 - 0 0 6.392836508 [EMIL1_MOUSE]Fibrillin-1 OS = Mus musculus GN = Fbn1 PE = 4 SV = 1 - 5.8777647595.704777353 0 [A2AQ53_MOUSE] Fibrinogen beta chain OS = Mus musculus GN= Fgb PE = 2 5.858490424 6.687289698 0 SV = 1 - [FIBB_MOUSE] Fibrinogengamma chain OS = Mus musculus GN = Fgg PE = 2 5.83914522 7.0408811196.623714799 SV = 1 - [FIBG_MOUSE] Fibrinogen, alpha polypeptide OS = Musmusculus GN = Fga 6.056325883 7.261485562 6.90940505 PE = 2 SV = 1 -[Q99K47_MOUSE] Fibronectin OS = Mus musculus GN = Fn1 PE = 1 SV = 4 -6.818688119 6.880740949 7.353539538 [FINC_MOUSE] Filamin, alpha(Fragment) OS = Mus musculus GN = Flna 7.008505708 6.6284361786.827984047 PE = 4 SV = 1 - [B7FAV1_MOUSE] Gelsolin OS = Mus musculus GN= Gsn PE = 1 SV = 3 - 5.729356743 5.763326472 0 [GELS_MOUSE] HaptoglobinOS = Mus musculus GN = Hp PE = 1 SV = 1 - 0 6.109791413 0 [HPT_MOUSE]Hemoglobin subunit alpha OS = Mus musculus GN = Hba 0 6.107901396.398904972 PE = 1 SV = 2 - [HBA_MOUSE] Hemopexin OS = Mus musculus GN =Hpx PE = 1 SV = 2 - 0 6.010280907 0 [HEMO_MOUSE] Histone H2A OS = Musmusculus GN = Hist1h2al PE = 2 SV = 1 - 7.392138835 6.9170800967.013660548 [F8WIX8_MOUSE] Histone H2A type 1-H OS = Mus musculus GN =Hist1h2ah 7.392138835 6.926879953 6.857002872 PE = 1 SV = 3 -[H2A1H_MOUSE] Histone H2B type 1-F/J/L OS = Mus musculus GN = Hist1h2bf7.491390907 7.124577253 7.005403699 PE = 1 SV = 2 - [H2B1F_MOUSE]Histone H3 (Fragment) OS = Mus musculus GN = H3f3a PE = 2 7.1014845146.959792034 7.049922513 SV = 1 - [E0CZ27_MOUSE] Histone H4 OS = Musmusculus GN = Hist1h4a PE = 1 SV = 2 - 7.676125661 7.5807810027.458167442 [H4_MOUSE] Ig gamma-1 chain C region secreted form OS = Musmusculus 5.961455553 0 0 GN = Ighg1 PE = 1 SV = 1 - [IGHG1_MOUSE] Igkappa chain V-II region 26-10 OS = Mus musculus PE = 1 6.4190145685.836803643 0 SV = 1 - [KV2A7_MOUSE] Ig mu chain C region secreted formOS = Mus musculus 6.201286175 6.434377902 6.526314192 GN = Igh-6 PE = 1SV = 2 - [IGHM_MOUSE] Indolethylamine N-methyltransferase OS = Musmusculus 5.637864703 0 0 GN = Inmt PE = 1 SV = 1 - [INMT_MOUSE] Junctionplakoglobin OS = Mus musculus GN = Jup PE = 1 6.264791243 6.4654100087.017601329 SV = 3 - [PLAK_MOUSE] Lactotransferrin OS = Mus musculus GN= Ltf PE = 2 SV = 4 - 0 5.762688986 0 [TRFL_MOUSE] Laminin subunitalpha-2 OS = Mus musculus GN = Lama2 5.937766596 0 0 PE = 2 SV = 1 -[F8VQ43_MOUSE] Laminin subunit alpha-3 OS = Mus musculus GN = Lama36.674990286 6.260528414 0 PE = 4 SV = 1 - [E9PUR4_MOUSE] Laminin subunitalpha-4 OS = Mus musculus GN = Lama4 6.511385176 6.099945813 0 PE = 1 SV= 2 - [LAMA4_MOUSE] Laminin subunit alpha-5 OS = Mus musculus GN = Lama56.607315399 6.547091642 6.327053775 PE = 1 SV = 4 - [LAMA5_MOUSE]Laminin subunit beta-1 OS = Mus musculus GN = Lamb1 5.951335808 0 0 PE =1 SV = 3 - [LAMB1_MOUSE] Laminin subunit beta-2 OS = Mus musculus GN =Lamb2 6.610256604 6.263976054 0 PE = 2 SV = 2 - [LAMB2_MOUSE] Lamininsubunit beta-3 OS = Mus musculus GN = Lamb3 6.687215625 6.379447773 0 PE= 2 SV = 2 - [LAMB3_MOUSE] Laminin subunit gamma-1 OS = Mus musculus GN= Lamc1 6.70043806 6.209442052 6.048843786 PE = 2 SV = 1 -[F8VQJ3_MOUSE] Laminin subunit gamma-2 OS = Mus musculus GN = Lamc26.218905096 6.142010192 0 PE = 4 SV = 1 - [G5E874_MOUSE] Lumican OS =Mus musculus GN = Lum PE = 1 SV = 2 - 6.189723414 0 0 [LUM_MOUSE]Lysozyme C-2 OS = Mus musculus GN = Lyz2 PE = 1 SV = 2 - 5.833915446.30672877 0 [LYZ2_MOUSE] MCG1050941 OS = Mus musculus GN = Gm5414 PE =2 SV = 1 - 6.899619386 7.13581107 7.369324207 [Q6IFZ8_MOUSE] MCG16555 OS= Mus musculus GN = Vdac3-ps1 PE = 4 SV = 1 - 5.516224232 0 0[J3QPE8_MOUSE] Microfibril-associated glycoprotein 4 OS = Mus musculus6.342904739 6.43463342 0 GN = Mfap4 PE = l SV = 1 - [MFAP4_MOUSE]Mimecan OS = Mus musculus GN = Ogn PE = 2 SV = 1 - 0 5.701058917 0[MIME_MOUSE] Myelin proteolipid protein OS = Mus musculus GN = Plp16.987098666 6.432634236 6.586369313 PE = 1 SV = 2 - [MYPR_MOUSE] Myeloidbactenecin (F1) OS = Mus musculus GN = Ngp PE = 2 5.44115199 6.1421010180 SV = 1 - [O08692_MOUSE] Myeloperoxidase OS = Mus musculus GN = Mpo PE= 2 SV = 2 - 0 6.382761391 0 [PERM_MOUSE] Myosin-10 OS = Mus musculus GN= Myh10 PE = 1 SV = 2 - 6.107244043 0 0 [MYH10_MOUSE] Myosin-11 OS = Musmusculus GN = Myh11 PE = 4 SV = 1 - 6.549138497 6.023621662 6.486852262[E9QPE7_MOUSE] Myosin-9 OS = Mus musculus GN = Myh9 PE = 1 SV = 4 -6.534077311 5.875775823 6.34610934 [MYH9_MOUSE] Neurofilament heavypolypeptide OS = Mus musculus 6.138902554 6.97864523 6.975120413 GN =Nefh PE = 1 SV = 3 - [NFH_MOUSE] Nidogen-1 OS = Mus musculus GN = Nid1PE = 1 SV = 2 - 7.128370461 7.113290014 6.730533502 [NID1_MOUSE]Nidogen-2 OS = Mus musculus GN = Nid2 PE = 1 SV = 2 - 6.2414084485.983593362 0 [NID2_MOUSE] Peptidyl-prolyl cis-trans isomerase B OS =Mus musculus 6.012874469 0 0 GN = Ppib PE = 2 SV = 2 - [PPIB_MOUSE]Periostin OS = Mus musculus GN = Postn PE = 1 SV = 2 - 6.7257252416.251665549 6.20505634 [POSTN_MOUSE] Peroxiredoxin-1 (Fragment) OS = Musmusculus GN = Prdx1 5.981037111 5.753060397 6.142601977 PE = 2 SV = 1 -[B1AXW5_MOUSE] Phosphate carrier protein, mitochondrial OS = Musmusculus 5.91683531 0 0 GN = Slc25a3 PE = 1 SV = 1 - [MPCP_MOUSE]Platelet glycoprotein 4 OS = Mus musculus GN = Cd36 6.163842655.950272926 0 PE = 1 SV = 2 - [CD36_MOUSE] Polyubiquitin-C (Fragment) OS= Mus musculus GN = Ubc 6.968911307 6.84889605 6.756181153 PE = 2 SV =1 - [E9Q5F6_MOUSE] Prelamin-A/C OS = Mus musculus GN = Lmna PE = 1 SV =2 - 5.749092722 0 0 [LMNA_MOUSE] Protein 4732456N10Rik OS = Mus musculusGN = 4732456N10Rik PE = 3 7.2526558 7.399852941 7.726082657 SV = 1 -[E9Q1Z0_MOUSE] Protein Col4a5 (Fragment) OS = Mus musculus GN = Col4a57.141558716 7.11299109 7.245885041 PE = 4 SV = 1 - [F7CK55_MOUSE]Protein Col4a6 OS = Mus musculus GN = Col4a6 PE = 2 SV = 1 - 6.6676635397.041405713 0 [B1AVK5_MOUSE] Protein Col6a3 OS = Mus musculus GN =Col6a3 PE = 4 SV = 1 - 7.182772976 6.887860833 6.651938048[J3QQ16_MOUSE] Protein Krt78 OS = Mus musculus GN = Krt78 PE = 2 SV =1 - 7.991649161 8.184126545 8.640017022 [E9Q0F0_MOUSE] Protein-glutaminegamma-glutamyltransferase 2 OS = Mus 6.606623968 6.34960286 6.828607255musculus GN = Tgm2 PE = 1 SV = 4 - [TGM2_MOUSE] Serotransferrin OS = Musmusculus GN = Tf PE = 1 SV = 1 - 5.569903604 5.983104216 0 [TRFE_MOUSE]Serum albumin OS = Mus musculus GN = Alb PE = 1 SV = 3 - 6.7698562177.211712096 6.555648421 [ALBU_MOUSE] Spectrin alpha chain,non-erythrocytic 1 OS = Mus musculus 6.273962184 0 0 GN = Sptan1 PE = 2SV = 1 - [A3KGU5_MOUSE] Spectrin beta chain, non-erythrocytic 1 OS = Musmusculus 5.993515702 0 0 GN = Sptbn1 PE = 1 SV = 2 - [SPTB2_MOUSE]Tenascin GRCm38.p3 [GCF_000001635.23] 7.12532211 6.112490357 6.730512501Titin OS = Mus musculus GN = Ttn PE = 2 SV = 1 - 5.743291257 6.2951324270 [E9Q8K5_MOUSE] Tubulin alpha-1C chain OS = Mus musculus GN = Tuba1c6.33172549 5.982744883 6.399140301 PE = 1 SV = 1 - [TBA1C_MOUSE]Tubulointerstitial nephritis antigen-like OS = Mus musculus 5.8314584840 0 GN = Tinagl1 PE = 2 SV = 1 - [H3BJ97_MOUSE] Voltage-dependent anion-selective channel protein 1 5.666833354 5.405315445 0 OS = Mus musculusGN = Vdac1 PE = 1 SV = 3 - [VDAC1_MOUSE] von Willebrand factor OS = Musmusculus GN = Vwf PE = 1 5.757768857 0 0 SV = 2 - [VWF_MOUSE]

Example 5 Inhibition of MTI-MMP Protects from Tissue Destruction WithoutModulating the Immune Response

MT1-MMP knockout mice suffer from multiple abnormalities and die within3-5 weeks after birth (Holmbeck et al., 1999); hence, the role ofMT1-MMP was evaluated using a selective allosteric inhibitory antibodyof MT1-MMP (LEM 2/15) effective at nano-molar concentrations (Udi etal., 2015). Importantly, this antibody has been shown to selectivelyinteract with MT1-MMP expressed on the cell surface and inhibit itscollagenase activity without significantly interfering with proMMP-2activation and enzyme dimerization on the cell surface (Udi et al.,2015). In order to evaluate the role of MT1-MMP during influenzainfection, mice were infected with sub-lethal doses of influenza virusand treated with either vehicle (PBS), control antibody or anti-MT1-MMPantibody during the course of the disease (26, 49, 74 hours; FIGS.4A-4K). Representative images are shown from tissue sections at 74 hourspost infection in FIGS. 4A-4K. AirSEM imaging of hydratedde-cellularized tissues demonstrated that blocking MT1-MMPcollagenolytic activity protected tissue integrity (both alveolar andbronchi structures), ECM morphology, and collagen structure as well asthe molecular composition of ECM scaffolds (FIGS. 4B-4E). 3D analysis oftwo-photon microscopy in second harmonic generation showed that collagentype I fibers were more abundant and maintained a continuous alveolarsac boundaries when infected mice were treated with an anti-MT1-MMPinhibitor (FIGS. 4F-4G). Furthermore, blocking MT1-MMP proteolyticactivity also protected laminin, a major basement-membrane constituent,from degradation in the infected lungs (FIGS. 4H-4J). Finally,functional in situ zymography indicated significant attenuation ofproteolysis following treatment (FIGS. 4J-4K), suggesting that MT1-MMPis a major driver of tissue destruction in the infection setting.

In order to better understand whether MT1-MMP is involved in immunemodulation, the abundance of macrophages, neutrophils, lymphocytes andnatural killer (NK) cells was analyzed at 74 hours post-infection usingeither anti-MT1-MMP inhibitor or a control antibody. The results showthat there is enhanced recruitment of both macrophages and neutrophilsto infected lungs with no detectable differences between anti-MT1-MMPand control-treated animals (FIG. 11A). In addition, in lung tissuesections stained for F4/80 marker, similar macrophage infiltration wasobserved upon anti-MT1-MMP treatment (FIGS. 11B-11C). Additionally,broncho-alveolar lavage fluid (BALF) was collected from bothanti-MT1-MMP treated as well as control treated mice in order toevaluate cytokine level of major immune-modulating cytokines (IL-1β andTNF-α), which have been shown to play a major role in the infectionprocess (Aldridge et al., 2009; Glaccum et al., 1997; Goldbach-Manskyand Kastner, 2009). Both IL-1β and TNF-α were strongly induced ininfluenza-infected mice regardless of MT1-MMP activity (FIGS. 11D-11F),suggesting the immune response was unaffected.

To evaluate whether MT1-MMP activity modulates viral loads duringinfluenza infection, the plaque forming unit (PFU) assay was carried out(FIG. 12A) and also the viral RNA was quantified using PFU assay (FIG.12B). In addition, lung tissue sections were stained for viral abundanceat both 24 and 74 hours post infection (FIGS. 12D-12F). Overall, theseanalyses showed minimal effect of MT1-MMP inhibition on viral burdenwhich was limited to the early phases of infection (24 hours); at laterstages, no effect was discernible. These results demonstrate thatMT1-MMP is not significantly involved in immune modulation or regulationof viral loads; thus, the major MT1-MMP influence on influenza infectionis the massive ECM fibrillary protein degradation and tissue damagestemming from its collagenase activity.

Example 6 Tissue Damage Results from Host Proteolytic Activity Ratherthan Viral Cytopathology

The present inventors then investigated whether the destructivephenotypes in the lung tissue are a direct consequence of viralcytopathology or rather a result of a host-associated immune responsedriving the dysregulated ECM proteolysis. The conventional influenzatreatment, Oseltamivir phosphate (Tamiflu), a selective inhibitor ofinfluenza A and B viral neuraminidase was analyzed (FIGS. 13A-13D).Virus titers from whole-lung homogenates of vehicle-treated andTamiflu-treated mice were quantified using qPCR (FIG. 13C) and comparedto topography of lung structural features visualized in lung tissuesections using AirSEM (FIGS. 9A-9B). Tamiflu dramatically reduces theviral burden (10-100 fold), meaning the tissue is exposed to low butpersistent viral presence (FIG. 13C). In spite of the lower viraltiters, the same destructive lung tissue and ECM phenotypes wereobserved, including multifocal alveolar wall thinning and a substantialloss of alveolar cells, in both vehicle-treated and Tamiflu-treated mice(FIG. 13A, 13B). Such irreversible destruction may be the main cause forloss of barrier integrity, and thus provides a window of opportunity forbacterial invasion. These results prompted the present inventors to testwhether protecting ECM integrity via blocking MT1-MMP proteolysis canimprove the lethal outcome of influenza-bacteria co-infections.

Example 7 Blocking MTI-MMP in Infected Mice Promotes Tissue Maintenanceand Prevents Sepsis

As noted above, the viral infection of influenza results in overwhelmingdamage to the ECM molecular composition and lung structure even withcurrent first-line influenza medication targeting the virus. To furthersupport the physiological consequences of influenza-induced ECMproteolysis and to mimic the frequent conditions that endangerinfluenza-infected hospitalized patients, an established protocol ofinfluenza secondary bacterial infection using S. pneumoniae was used(McCullers and Herman, 2001; McCullers, 2003; McCullers, 2004). 10groups of mice (10 mice per group) were infected twice within a range of96 hours combining PR8 influenza virus followed by S. pneumoniae, withboth at sub-lethal doses (FIGS. 5A-5H), and the different groups weretreated with either vehicle, irrelevant control antibody, Tamiflu,anti-MT1-MMP, or a combined therapy of both agents. To mimic potentialtreatment modes, mice were treated in two different protocols;prophylactic (before the infection) or therapeutic (post infection)(FIGS. 5A-5B). The group of mice that was treated with Tamiflu as apreventive agent (Tamiflu-1) exihibited the same survival rates andclinical scores as the group that received anti-MT1-MMP (FIGS. 5B-5C),suggesting that anti-viral or ECM protection are equally effective inpreventing co-infection induced mortality when administered as apreventive treatment.

In line with reports (Jefferson et al., 2014) demonstrating that Tamifluis not effective in reducing hospitalozation duration or influenzasymptoms when administered post-infection, co-infected mice treated withTamiflu (+1 group) did not exhibit an improved response when comparedwith the vehicle group (20% survival) (FIGS. 5E-5F). In these sametherapeutic settings (FIG. 5D), treatment with the anti-MT1-MMPinhibitor was significantly better, indicating a pronounced theraputiceffect (70% survival). Importantly, combined application of Tamiflu andanti-MT1-MMP treatment resulted in 100% survival rates when usedpreventatively or therapeutically (FIGS. 5B-5E). This was furthersupported by AirSEM images demonstrating the destructive phenotype ofalveolar and bronchial structures of the Tamiflu (−1) group, whichsuggest that even as a prophylactic measure, Tamiflu is ineffective inpreventing collateral damage in the lung ECM (FIGS. 14A-14B). To extendthese observations of ECM fibrillar protein degradation, destruction ofbasement membrane constituents and disruption of the air-blood barrierin influenza infection, the present inventors further tested forbacterial dissemination into the blood stream (sepsis) and infections indistant organs of co-infected mice. It was found that vehicle-treatedmice developed bacteremia and show dissemination of S. pneumoniae intothe spleen 2 days post bacterial infection, while mice receivinganti-MT1-MMP inhibitor did not develop systemic bacterial disseminationand maintained a local and confined lung infection (FIGS. 5G-5H).

Discussion

The present examples suggest that therapeutic strategies to fightinfluenza infections should be aimed not only at the viral infectivitybut also with the purpose of increasing tissue tolerance. This isespecially true when taking into consideration that secondary bacterialinfections involving S. pneumoniae, Staphylococcus aureus, andHaemophilus influenzae among others that are the main risk factors forreduced survival among high-risk populations (McCullers and Herman,2001; McCullers, 2014; Morens et al., 2008). Co-infections, in manycases, involve severe lung inflammation driven by robust responses ofthe immune system to the viral insult. These responses dramaticallyimpair tissue homeostasis, including disruption of the respiratoryepithelium, ECM and basement membrane elements. The host response toviral infections includes tissue damage associated with oxidative stress(Avissar et al., 1996; Bozinovski et al., 2012; Campbell et al., 1987;Suliman et al., 2001; Yatmaz et al., 2013). On the one hand, MMPactivity is required for the normal immune response to infection. On theother hand, host-derived MMPs may also cause infection-relatedimmunopathology. This paradox results from the delicate balance betweennormal MMP function and destructive MMP-related host tissue damage(Elkington et al., 2005). Since MMPs play a crucial role in irreversibleremodeling of the ECM, these robust proteases are tightly controlled andregulated (Gaffney J, 2015; Lopez-Otin and Matrisian, 2007; Turk, 2006).Moreover, maintaining tissue homeostasis during infection can bechallenging when immune cells expressing active proteases are recruitedtowards respiratory pathogens.

Using genome-wide transcriptional profiling of influenza infected lungs,an extraordinarily large number of genes engaged in extracellular matrixturnover and protein catabolism were observed at various time pointsduring the infection course. Previous studies have assessed thetranscriptional signatures during influenza infection by comparing invitro host responses to different influenza viral strains using humanrespiratory bronchial and epithelial cell lines as well as in vivoexperiments in mice and ferrets (Bortz et al., 2011; Brandes M, 2013;Chevrier et al., 2011; Elkington et al., 2005; Hartmann et al., 2015;Josset et al., 2014; Kash et al., 2004; Leon et al., 2013; Ljungberg etal., 2012; Peng et al., 2014; Shapira et al., 2009). To evaluate therelevance of MT1-MMP to human infections, data from primary humanbronchial epithelial cells infected in vitro with a H1N1 influenzastrain A/PR/8/34 was analyzed. While human macrophages would be a bettermodel, this data show that MT1-MMP is up-regulated also in a primaryhuman cell model (FIG. 15) (Shapira et al., 2009) suggesting therelevance of our findings to the human ECM remodeling response toinfluenza infection.

Expanding on these studies, the present inventors focused a systematicanalysis on ECM remodeling and, specifically, the activity of MT1-MMPduring influenza infection. Noticeably, it was found that MT1-MMP isexpressed almost entirely by stromal cells in the healthy lung duringhomeostasis. In contrast, following infection, MT1-MMP expression isprimarily observed in the immune compartment and was accompanied byincreased collagenolytic activity. Analysis of MT1-MMP-expressing cellpopulations showed a robust relationship with cytokine, chemokines andanti-viral response genes confirming that MT1-MMP is an inherent circuitof the host anti-viral response program. It was also found that multipleknown substrates of MT1-MMP (Koziol Al, 2012; Stegemann et al., 2013),including fibrillary and basement membrane collagens (colIV, colXII,colXIV) as well as proteoglycans, are irreversibly cleaved and lost fromthe lungs of influenza-infected mice. These compositional changes wereaccompanied by ECM scaffold degradation and depletion of epithelialcells, thus contributing to a destructive phenotype that included lossof alveolar space, thinning of the alveolar wall and distortion ofairway structures. Together, it has been shown that influenza infectionsinduce expression and activity of MT1-MMP, which contributes touncontrolled degradation of the structural ECM components that arerequired for maintaining the lung integrity and function.

Elevated MT1-MMP expression levels have been described in the context ofcancer cell metastasis implicating MT1-MMP as an invasive markerassociated with advanced stages and poor prognosis (Zarrabi et al.,2011). This property was attributed to the collagenolytic activity ofMT1-MMP, degrading the peri-cellular environment and endothelialbarriers to make a path for metastatic cells. However, the role ofMT1-MMP in influenza infection has not been previously reported.Accordingly, elevated expression levels of tissue inhibitors ofmetalloproteinases (TIMPs), such as TIMP-1 and TIMP-3, the endogenousinhibitors of most metalloproteinases were noted. Nevertheless,expression levels of TIMP-2 the endogenous inhibitor of MT1-MMP were notsignificantly changed. Other ECM enzymes have been shown to play a rolein influenza infection as well (Bradley et al., 2012). A significantincrease in MMP-8 at the protein level was also noted (FIGS. 6A-6D).

Previous studies have sought to disentangle viral cytopathic effectsfrom inflammatory collateral damage during influenza infection (Boon etal., 2011; Kash et al., 2004; Kobasa et al., 2007; Tate et al., 2009).It has now been shown that even in low viral titers, lung ECMdestruction is significant. This suggests that the main damage to theECM during infection is a parallel pathway driven by proteolytic eventsassociated with host response irrespective of direct viral loads(Jamieson et al., 2013; Medzhitov et al., 2012; Schneider and Ayres,2008). Under these conditions, it has now been shown that ECM damage canbe almost completely rescued by selectively modulating MT1-MMPproteolytic activity. Using an anti-MT1-MMP inhibitory Fab fragment, itwas possible to maintain tissue structure and improve the outcome ofinfluenza infections irrespective of viral replication. It is noteworthythat this MT1-MMP antibody is highly selective in targeting thecollagenase activity and does not interfere with enzyme dimerization andmaturation of pro-MMP-2 required for tissue homeostasis (Udi et al.,2015). The present study shows that MT1-MMP is not critically involvedin immune cell recruitment or IL-1β and TNF-α production. This is inline with previous studies showing that macrophage-derived MT1-MMPregulated subjacent cellular proteolysis rather than directly beinginvolved in migration or cell trafficking through host tissues(Shimizu-Hirota et al., 2012).

In order to mimic the natural disease progression, co-infection settingsof influenza and S. pneumoniae were used to show that targeting thevirus with Tamiflu alone is ineffective in controlling ECM damage anddoes not predict successful management of the disease followingbacterial infection. Importantly, inhibition of MT1-MMP activity, whichprotected tissue architecture and composition without significantlyaffecting the viral loads, exhibited improved disease management whenadministrated as either a prophylactic or therapeutic agent. Inagreement, mice treated with Tamiflu developed sepsis due todissemination of bacteria from the lungs to the systemic circulation,while those treated with the MT1-MMP inhibitory antibody exhibitedreduced spread of bacteria through blood-air disruption. This furthersuggests that the maintenance of tissue homeostasis is a parallelprocess that, at least in our influenza model, is as importanttherapeutically as controlling the viral load. Importantly, thecombination of the two treatments achieved complete survival rates bothin the prophylactic and therapeutic modes. This further supports thepresent findings that the combination of the two strategies, targetingviral replication as well as maintaining host barrier homeostasis andpreventing tissue destruction, greatly increases the survival outcome.

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Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting. In addition, any priority document(s) of this applicationis/are hereby incorporated herein by reference in its/their entirety.

What is claimed is:
 1. A method of treating a respiratory infection in asubject in need thereof comprising administering to the subject atherapeutically effective amount of an agent which down-regulates anextracellular matrix-associated polypeptide selected from the groupconsisting of membrane type 1-matrix metalloproteinase 1 (MT1-MMP1),MMP-9, MMP-8 and MMP-3, thereby treating the respiratory infection. 2.The method of claim 1, wherein said agent is an antibody.
 3. The methodof claim 1 wherein said extracellular matrix-associated polypeptide isMT1-MMP1.
 4. The method of claim 1, wherein the administering iseffected no more than 2 days after the start of symptoms of theinfection.
 5. The method of claim 1, wherein the infection is a viralinfection.
 6. The method of claim 1, wherein said infection is an upperrespiratory tract infection.
 7. The method of claim 1, wherein saidinfection is a lower respiratory tract infection.
 8. The method of claim5, further comprising administering to the subject an anti-viral agent.9. The method of claim 8, wherein said anti-viral agent is aneuraminidase inhibitor (NAI).
 10. The method of claim 9, wherein saidneuraminidase inhibitor is selected from the group consisting ofLaninamivir, Oseltamivir, Peramivir and Zanamivir.
 11. The method ofclaim 3, wherein said agent is an antibody that targets residues 160-173and/or residues 218-233 of said MT1-MMP1.
 12. The method of claim 3,wherein said agent is an antibody that down-regulates the collagenaseactivity of said MT1-MMP1.
 13. A method of preventing a diseaseassociated with a secondary infection in a subject infected with apathogen comprising administering to the subject a therapeuticallyeffective amount of an anti-pathogenic agent directed towards saidpathogen and a therapeutically effective amount of an agent whichdown-regulates at least one extracellular matrix-associated polypeptide,thereby preventing the disease associated with a secondary infection inthe subject.
 14. The method of claim 13, wherein said extracellularmatrix-associated polypeptide is set forth in Table
 1. 15. The method ofclaim 13, wherein said secondary infection is a blood infection.
 16. Themethod of claim 13, wherein said disease is sepsis.
 17. The method ofclaim 13, wherein said extracellular matrix-associated polypeptide isselected from the group consisting of membrane type 1-matrixmetalloproteinase 1 (MT1-MMP1), MMP-9, MMP-8 and MMP-3.
 18. The methodof claim 13, wherein said virus is a respiratory virus.
 19. The methodof claim 13, wherein said agent which down-regulates said at least onepolypeptide is an antibody.